Biochimica Sistematica

Umana

HUMAN SYSTEMATIC BIOCHEMISTRY

(6 cfu)

Gianluca Paventi

e-mail: gianluca.paventi@unifg.it
Tel:
Skype account: gianluca.paventi
 OBIETTIVI

Fornire, attraverso un approccio didattico atto a favorire lo

sviluppo/il consolidamento di capacità critica e metodo
scientifico, gli strumenti per comprendere, a livello biochimico e
molecolare, i complessi fenomeni di comunicazione tra organi e
tessuti, i sistemi di controllo delle loro funzioni e le loro
interrelazioni in condizioni fisiologiche.
 Testi consigliati:

• Biochimica Sistematica Umana - Caldarera - Clueb

• Biochimica Medica - Strutturale, metabolica e funzionale - Siliprandi &Tettamanti - Piccin

• Biochimica con aspetti clinici - T. M. Devlin-Idelson-Gnocchi

• Fondamenti di biochimica umana – M. Maccarone - Zanichelli

• Biochimica umana – F. Salvatore- Idelson Gnocchi

The uses of Biochemistry:

1. Agriculture
2. Medicine
3. Nutrition
4. Clinical chemistry
5. Pharmacology
6. Toxicology
7. …
• “Living things are composed of lifeless
molecules” (Albert Lehninger)

• “Chemistry is the logic of biological

phenomena” (Garrett and Grisham)
“…mi sono dato come legge di procedere
sempre dal noto all’ignoto, di non fare
alcuna deduzione che non sgorgasse
direttamente dagli esperimenti e dalla
osservazione, … di non colmare mai il
silenzio dei fatti con affrettate conclusioni”

A.L. Lavoisier (1743-1794)

 Per trattare un qualunque
argomento è fondamentale la

DEFINIZIONE
 BIOCHIMICA

bio = vita…
chimica della vita
Definizione in termini di
STRUTTURA

enzima
catalizzatore
proteina

Definizione in termini di
FUNZIONE
Struttura e funzione sono
aspetti inseparabili
proteine

membrane
fondamentale ricordare:

CHI E’
E
QUANTO CE N’E’!
Principio dell’equilibrio mobile

Ogni sistema, sottoposto a

variazione, si oppone ad essa
 Principio di economia

Ogni sistema biologico opera alla

luce del principio di massima
economia di parti e processi
 La biochimica è una scienza
Empirico, dal greco empirikos, composto da en- ("che
empirica si muove nella"), peira ("esperienza"). Empirico è ciò
che si muove entro l'esperienza dei fenomeni, ciò che
è pratico e fa riferimento ai soli accadimenti e
fenomeni che giungono ai sensi

riduzionista Comprendere i principi chimici e fisici alla base della

vita. Per far ciò a volte è necessario scomporre
processi complessi in unità più semplici per studiarli
meglio: es. studiare un organello subcellulare isolato
dalla cellula, una tappa di una via metabolica etc…
 Gli organismi viventi
obbediscono alle leggi
della fisica e della
chimica
Le reazioni biochimiche
richiedono una catalisi

enzimi
 Distinctive Properties of Living
Systems

• Organisms are complicated and highly organized

• Biological structures serve functional purposes
• Living systems are actively engaged in energy
transformations
• Living systems have a remarkable capacity for
self-replication
Chemically usefull form of energy stored
compartimentazione
 Simple Molecules are the Units for
Building Complex Structures
• Metabolites and Macromolecules
• Organelles
• Membranes
• The Unit of Life is the Cell
 Properties of Biomolecules
Reflect Their Fitness to the
Living Condition
Important numbers!
• van der Waals: 0.4-4.0 kJ/mole
• Hydrogen bonds: 12-30 kJ/mole
• Ionic bonds: 20 kJ/mole
• Hydrophobic interactions: <40 kJ/mole
Legame Idrogeno
 Two Important Points About
Weak Forces

• Biomolecular Recognition is Mediated by

Weak Chemical Forces

• Weak Forces Restrict Organisms to a

Narrow Range of Environmental
Conditions
 Water, pH, and Ionic Equilibria

All rights reserved. Requests for permission to make copies of any part of the work
should be mailed to: Permissions Department, Harcourt Brace & Company, 6277
Sea Harbor Drive, Orlando, Florida 32887-6777
 Outline
• Properties of Water
• pH
• Buffers
• Water's Unique Role in the Fitness of the
Environment
 Properties of Water
• High b.p., m.p., heat of vaporization,
surface tension
• Polar structure
• Non-tetrahedral bond angles
• H-bond donor and acceptor
• Potential to form four H-bonds per water
 Solvent Properties of Water
• Ions are always hydrated in water and
carry around a "hydration shell“

• Water forms H-bonds with polar solutes

• Hydrophobic interactions - a "secret of life"

 Hydrophobic Interactions

• A nonpolar solute "organizes" water

• The H-bond network of water reorganizes
to accommodate the nonpolar solute
• This is an increase in "order" of water
• This is a decrease in ENTROPY
 Amphiphilic Molecules
Also called "amphipathic"
• Refers to molecules that contain both
polar and non-polar groups
• Equivalently - to molecules that are
attracted to both polar and non-polar
environments
• Good examples - fatty acids
The Cell Membrane and Cell Transport
Functions of Cell Membranes
1. Separate cell from nonliving environment. Form
most organelles and partition cell into discrete
compartments.
2. Regulate passage of materials in and out of the
cell and organelles. Membrane is selectively
permeable.
3. Receive information that permits cell to sense
and respond to environmental changes.
! Hormones
! Growth factors
! Neurotransmitters
4. Communication with other cells and the
organism as a whole. Surface proteins allow cells
to recognize each other, adhere, and exchange
materials.
I. Fluid Mosaic Model of the Membrane
1. Phospholipid bilayer: Major component is a
phospholipid bilayer.
! Hydrophobic tails face inward
! Hydrophilic heads face water
2. Mosaic of proteins: Proteins “float” in the
phospholipid bilayer.
3. Cholesterol: Maintains proper membrane
fluidity.
The outer and inner membrane surfaces are
different.
Membrane
Phospholipids
Form a
Bilayer
 The Membrane is a Fluid Mosaic of
Phospholipids and Proteins

Notice that inner and outer surfaces are different

A. Fluid Quality of Plasma Membranes

! In a living cell, membrane has same fluidity as

salad oil.
! Unsaturated hydrocarbon tails INCREASE membrane
fluidity
! Phospholipids and proteins drift laterally.
! Phospholipids move very rapidly
! Proteins drift in membrane more slowly
! Cholesterol: Alters fluidity of the membrane
! Decreases fluidity at warmer temperatures (> 37oC)
! Increases fluidity at lower temperatures (< 37oC)
B. Membranes Contain Two Types of Proteins
1. Integral membrane proteins:
Inserted into the membrane.
Hydrophobic region is adjacent to hydrocarbon tails.
2. Peripheral membrane proteins:
Attached to either the inner or outer membrane surface.
Functions of Membrane Proteins:
1. Transport of materials across membrane
2. Enzymes
3. Receptors of chemical messengers
4. Identification: Cell-cell recognition
5. Attachment:
! Membrane to cytoskeleton
! Intercellular junctions
Membrane Proteins Have Diverse Functions
The cell plasma membrane is Selectively Permeable
A. Permeability of the Lipid Bilayer
1. Non-polar (Hydrophobic) Molecules
• Dissolve into the membrane and cross with ease
• The smaller the molecule, the easier it can cross
• Examples: O2 , hydrocarbons, steroids

2. Polar (Hydrophilic) Molecules

• Small polar uncharged molecules can pass through
easily (e.g.: H2O , CO2)
• Large polar uncharged molecules pass with
difficulty (e.g.: glucose)

3. Ionic (Hydrophilic) Molecules

• Charged ions or particles cannot get through
(e.g.: ions such as Na+ , K+ , Cl- )
Transport Proteins in the membrane:
Integral membrane proteins that allow for
the transport of specific molecules across
the phospholipid bilayer of the plasma
membrane.
How do they work?
! May provide a “hydrophilic tunnel” (channel)
! May bind to molecule and physically move it

! Are specific for the atom/molecule transported

III. Passive transport: Diffusion of molecules
across the plasma membrane
A. Diffusion: The net movement of a substance
from an area of high concentration to area of
low concentration.
Does not require energy.
B. Passive transport: The diffusion of substance
across a biological membrane.
! Only substances which can cross bilayer by
themselves or with the aid of a protein
! Does not require the cell’s energy
Passive Transport: Diffusion Across a
Membrane Does Not Require Energy
IV. Osmosis:
The diffusion of water across a semi-permeable
membrane.
Through osmosis water will move from an area
with higher water concentration to an area with
lower water concentration.
Solutes can’t move across the semi-permeable
membrane.
Osmotic Pressure: Ability of a solution to take up water
through osmosis.
Example: The cytoplasm of a cell has a certain osmotic
pressure caused by the solutes it contains.
There are three different types of solution when compared to
the interior (cytoplasm) of a cell:
1. Hypertonic solution: Higher osmotic pressure than cell due to:
Higher solute concentration than cell or
Lower water concentration than cell.
2. Hypotonic solution: Lower osmotic pressure than cell due to:
Lower solute concentration than cell or
Higher water concentration than cell.
3. Isotonic solution: Same osmotic pressure than cell.
Equal concentration of solute(s) and water than cell.
V. Cells depend on proper water balance
Animal Cells:
Do best in isotonic solutions.
Examples:
! 0.9% NaCl (Saline)
! 5% Glucose
If solution is not isotonic, cell will be affected:
! Hypertonic solution: Cell undergoes crenation.
Cell “shrivels” or shrinks.
! Example: 5% NaCl or 10% glucose
! Hypotonic solution: Cell undegoes lysis. Cell
swells and eventually bursts.
! Example: Pure water.
V. Cells depend on proper water balance
Plant Cells: Do best in hypotonic solutions, because
the cell wall protects from excessive uptake of
water.
! Hypertonic solution: Cell undergoes plasmolysis.
Cell membrane shrivels inside cell wall.
! Isotonic solution: Cell becomes flaccid or wilts.
! Hypotonic solution: Turgor. Increased firmness of
cells due to osmotic pressure.
! This is the reason why supermarkets spray fruits and
vegetables with pure water, making them look firm and
fresh.
VI. Facilitated Diffusion:
Some substances cannot cross the membrane by
themselves due to their size or charge.
Membrane proteins facilitate the transport of solutes
down their concentration gradient.
No cell energy is required.

Transport Proteins
! Specific : Only transport very specific molecules
(binding site)
! Glucose
! Specific ions (Na+, K+, Cl- )
Facilitated Diffusion Uses a Membrane
Transport Protein
VI. Active Transport:
" Proteins use energy from ATP to actively “pump”
solutes across the membrane
" Solutes are moved against a concentration gradient.
" Energy is required.

Example:
The Na+-K+ ATPase pump:
Energy of ATP hydrolysis is used to move
Na+ out of the cell and K+ into the cell
 Dissociation of Weak
Electrolytes

Consider a weak acid, HA

• The acid dissociation constant is given by:
• HA ® H+ + A-
• Ka = [ H + ] [ A - ]
____________________

[HA]
 The Henderson-Hasselbalch
Equation

Know this! You'll use it constantly.

• For any acid HA, the relationship between the
pKa, the concentrations existing at equilibrium
and the solution pH is given by:
¯

[ A- ]
pH = pKa + log
[ HA]
 Buffers
• Buffers are solutions that resist changes in
pH as acid and base are added
• Most buffers consist of a weak acid and its
conjugate base
• Buffers can only be used reliably within a
pH unit of their pKa
Lipids & Membranes
Lipids are non-polar (hydrophobic) compounds,
soluble in organic solvents.
Most membrane lipids are amphipathic, having a
non-polar end and a polar end.
Fatty acids consist of a hydrocarbon chain with a
carboxylic acid at one end.
A 16-C fatty acid: CH3(CH2)14-COO-
Non-polar polar

A 16-C fatty acid with one cis double bond between

C atoms 9-10 may be represented as 16:1 cis D9.
 O
Double bonds in fatty b
g a C
acids usually have the 4
3 1 O
-
2
cis configuration.
Most naturally fatty acid with a cis-D9
occurring fatty acids double bond
have an even number
of carbon atoms.
Some fatty acids and their common names:
14:0 myristic acid; 16:0 palmitic acid; 18:0 stearic acid;
18:1 cisD9 oleic acid
18:2 cisD9,12 linoleic acid
18:3 cisD9,12,15 a-linonenic acid
20:4 cisD5,8,11,14 arachidonic acid
20:5 cisD5,8,11,14,17 eicosapentaenoic acid (an omega-3)
 O
b
g a C
4
3 1 O-
2

fatty acid with a cis-D9

double bond

There is free rotation about C-C bonds in the fatty acid

hydrocarbon, except where there is a double bond.
Each cis double bond causes a kink in the chain.
Rotation about other C-C bonds would permit a more
linear structure than shown, but there would be a kink.
 Glycerophospholipids
Glycerophospholipids
CH2OH
(phosphoglycerides), are common
constituents of cellular membranes. H C OH
They have a glycerol backbone.
CH2OH
Hydroxyls at C1 & C2 are esterified
to fatty acids. glycerol

An ester forms
when a hydroxyl Formation of an ester:
reacts with a
O O
carboxylic acid,
with loss of H2O. R'OH + HO-C-R" R'-O-C-R'' + H2O
 Phosphatidate
O

O H2C O C R2

R1 C O CH O

H2C O P O-

O-
phosphatidate

In phosphatidate:
w fatty acids are esterified to hydroxyls on C1 & C2
w the C3 hydroxyl is esterified to Pi.
 O

O H2C O C R2
Each glycerophospholipid
R1 C O CH O
includes
w a polar region: H2C O P O X
glycerol, carbonyl O O-
of fatty acids, Pi, & the glycerophospholipid
polar head group (X)
polar
w non-polar hydrocarbon
tails of fatty acids (R1, R2). "kink" due to
double bond non-polar
 O

O H2C O C R2

R1 C O CH O

H2C O P O X

O-
glycerophospholipid

In most glycerophospholipids (phosphoglycerides),

Pi is in turn esterified to OH of a polar head group (X):
e.g., serine, choline, ethanolamine, glycerol, or inositol.
The 2 fatty acids tend to be non-identical. They may differ
in length and/or the presence/absence of double bonds.
 O

O H2C O C R2

R1 C O CH O CH3
+
H2C O P O CH2 CH2 N CH3

O- CH3

phosphatidylcholine

Phosphatidylcholine, with choline as polar head

group, is another glycerophospholipid.
It is a common membrane lipid.
lecitine
Dipalmitoil lecitina > 80% dei fosfolipidi Maggior componente
che rivestono lo strato di acqua extracellulare
negli alveoli polmonari

Surfattante polmonare

inspirazione

atelectasia

espirazione

Alveoli normali
 O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H

OH OH
H OH
OH H
phosphatidyl- H H
inositol
H OH

Phosphatidylinositol, with inositol as polar head group,

is one glycerophospholipid.
In addition to being a membrane lipid,
phosphatidylinositol has roles in cell signaling.
 Prodotti dai leucociti:
contrazione della muscolatura
liscia (attacchi asmatici e
Prodotte da quasi tutte le allergie)
cellule: mediatori naturali
dei processi infiammatori
Prodotti dalle piastrine e coinvolti nella formazione di coaguli
 Ether Glycerophospholipids
An ether instead of an acyl group at C-1

Plasmalogens are ether

glycerophospholipids in which the alkyl
chain is unsaturated
 Ether Glycerophospholipids
• Platelet activating factor (PAF) is an ether
glycerophospholipid
• PAF is a potent biochemical signal
molecule
• Note the short (acetate) fatty acyl chain at
the C-2 position in PAF
Molto abbondanti nel
tessuto nervoso (mielina)
cardiaco (50% degli
etanolammina lipidi)

Membrane di cellule
cancerose
metastatiche
presentato un
elevato contenuto
eteroglicerolipidi
Mediatore:
• Ipersensibilità
• Reazioni infiammatorie
acute
• Shock anafilattico
• Reazioni allergiche

Sintetizzato e rilasciato in seguito

al legame con antigene delle IgE
presenti su macrofagi e leucociti
e monociti, determinando:
• Aggregazione piastrinica
• Edema
• Ipotensione
• Chemiotassi
 OH OH
H
H2C C CH
Sphingolipids are derivatives of the
lipid sphingosine, which has a long H3N+ CH
hydrocarbon tail, and a polar domain
HC
that includes an amino group.
(CH2 )12
OH OH sphingosine CH3
H
H2C C CH
The amino group of sphingosine can
NH CH form an amide bond with a fatty acid
O C HC
carboxyl, to yield a ceramide.
Ceramides usually include a polar
R (CH2 )12
head group, esterified to the terminal
ceramide CH3 OH of the sphingosine.
 Sphingolipid Biosynthesis

High levels made in neural tissue

• Initial reaction is a condensation of serine and
palmitoyl-CoA
• 3-ketosphinganine synthase is PLP-dependent
• Ketone is reduced with help of NADPH
• Acylation is followed by double bond formation
• Resulting ceramide is precursor for other
sphingolipids
 Sphingolipids
Base structure is sphingosine
• Sphingosine is an 18-carbon amino
alcohol
• Ceramides are amide linkages of fatty
acids to the nitrogen of sphingosine
• Glycosphingolipids are ceramides with
one or more sugars in beta-glycosidic
linkage at the 1-hydroxyl group
 CH3 O
H2 H2 -
+
H3C N C C O P O

CH3 O OH

Sphingomyelin, a phosphocholine H
H2C C CH
ceramide with a sphingosine NH CH
phosphocholine or
phosphethanolamine O C HC
head group, is a fatty acid R (CH2 )12
common constituent Sphingomyelin CH3
of plasma membranes

Sphingomyelin, with a phosphocholine head group, is

similar in size and shape to the glycerophospholipid
phosphatidyl choline.
 Sphingolipids
• Glycosphingolipids with one sugar are
cerebrosides
• Gangliosides - ceramides with 3 or more
sugars, one of which is a sialic acid
 CH2OH

OH O
H OH
OH H O
H
H H H2C C CH

H OH NH CH

O C HC

R (CH2 )12
cerebroside with
b-galactose head group CH3

A cerebroside is a sphingolipid (ceramide) with

a monosaccharide such as glucose or galactose
as polar head group.
A ganglioside is a ceramide
with a polar head group that is
a complex oligosaccharide,
including the acidic sugar
derivative sialic acid.
Cerebrosides and
gangliosides, collectively
called glycosphingolipids,
are commonly found in the
outer leaflet of the plasma
membrane bilayer, with their
sugar chains extending out
from the cell surface.
 O H

H3C C NH O COO-
R HC OH
H H R=
HC OH
H OH
CH2OH
OH H
N-acetylneuraminate (sialic acid)

N-acetylneuraminate (N-acetylneuraminic acid, also

called sialic acid) is often found as a terminal residue
of oligosaccharide chains of glycoproteins.
Sialic acid imparts negative charge to glycoproteins,
because its carboxyl group tends to dissociate a proton
at physiological pH, as shown here.
Amphipathic lipids in
association with water form
complexes in which polar
regions are in contact with
water and hydrophobic Bilayer Spherical Micelle
regions away from water.
Depending on the lipid, possible molecular arrangements:
w Various micelle structures. E.g., a spherical micelle is
a stable configuration for amphipathic lipids with a
conical shape, such as fatty acids.
w A bilayer. This is the most stable configuration for
amphipathic lipids with a cylindrical shape, such as
phospholipids.
Membrane fluidity:
The interior of a lipid bilayer
is normally highly fluid. liquid crystal crystal
In the liquid crystal state, hydrocarbon chains of
phospholipids are disordered and in constant motion.
At lower temperature, a membrane containing a single
phospholipid type undergoes transition to a crystalline
state in which fatty acid tails are fully extended, packing
is highly ordered, & van der Waals interactions between
adjacent chains are maximal.
Kinks in fatty acid chains, due to cis double bonds,
interfere with packing in the crystalline state, and lower
the phase transition temperature.
Cholesterol, an
important constituent
of cell membranes,
has a rigid ring
system and a short HO
branched Cholesterol
hydrocarbon tail.

Cholesterol is largely
hydrophobic.
But it has one polar group,
a hydroxyl, making it
amphipathic.

PDB 1N83 cholesterol

 Cholesterol
in membrane

Cholesterol inserts into bilayer membranes with its

hydroxyl group oriented toward the aqueous phase &
its hydrophobic ring system adjacent to fatty acid
chains of phospholipids.
The OH group of cholesterol forms hydrogen bonds
with polar phospholipid head groups.
Interaction with the relatively rigid
cholesterol decreases the mobility of Cholesterol
hydrocarbon tails of phospholipids. in membrane

But the presence of cholesterol in a phospholipid

membrane interferes with close packing of fatty acid
tails in the crystalline state, and thus inhibits transition
to the crystal state.
Phospholipid membranes with a high concentration of
cholesterol have a fluidity intermediate between the
liquid crystal and crystal states.
Two strategies by which phase changes of membrane
lipids are avoided:
w Cholesterol is abundant in membranes, such as
plasma membranes, that include many lipids with
long-chain saturated fatty acids.
In the absence of cholesterol, such membranes would
crystallize at physiological temperatures.
w The inner mitochondrial membrane lacks cholesterol,
but includes many phospholipids whose fatty acids
have one or more double bonds, which lower the
melting point to below physiological temperature.
 Testi consigliati:

• Biochimica Sistematica Umana - Caldarera - Clueb

• Biochimica Medica - Strutturale, metabolica e funzionale -

Siliprandi &Tettamanti - Piccin

• Biochimica con aspetti clinici - T. M. Devlin-Idelson-Gnocchi

• Fondamenti di biochimica umana – M. Maccarone - Zanichelli

• Biochimica umana – F. Salvatore- Idelson Gnocchi

VI. Active Transport:
! Proteins use energy from ATP to actively “pump”
solutes across the membrane
! Solutes are moved against a concentration gradient.
! Energy is required.

Example:
The Na+-K+ ATPase pump:
Energy of ATP hydrolysis is used to move
Na+ out of the cell and K+ into the cell
 O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H

OH OH
H OH
OH H
phosphatidyl- H H
inositol
H OH

Phosphatidylinositol, with inositol as polar head group,

is one glycerophospholipid.
In addition to being a membrane lipid,
phosphatidylinositol has roles in cell signaling.
 Prodotti dai leucociti:
contrazione della muscolatura
liscia (attacchi asmatici e
Prodotte da quasi tutte le allergie)
cellule: mediatori naturali
dei processi infiammatori
Prodotti dalle piastrine e coinvolti nella formazione di coaguli
 Cholesterol
in membrane

Cholesterol inserts into bilayer membranes with its

hydroxyl group oriented toward the aqueous phase &
its hydrophobic ring system adjacent to fatty acid
chains of phospholipids.
The OH group of cholesterol forms hydrogen bonds
with polar phospholipid head groups.
Composizione lipidica membrane
Lateral mobility of a lipid,
within the plane of a
membrane, is depicted at
right and in an animation. Lateral Mobility
High speed tracking of individual lipid molecules has
shown that lateral movements are constrained within
small membrane domains.
Hopping from one domain to another occurs less
frequently than rapid movements within a domain.
The apparent constraints on lateral movements of lipids
(and proteins) has been attributed to integral membrane
proteins, anchored to the cytoskeleton, functioning as a
picket fence.
 peripheral
Membrane lipid
proteins may be anchor
classified as:
w peripheral
lipid bilayer
w integral
w having a Membrane
lipid anchor integral Proteins
Peripheral proteins are on the membrane surface.
They are water-soluble, with mostly hydrophilic surfaces.
Often peripheral proteins can be dislodged by conditions
that disrupt ionic & H-bond interactions, e.g., extraction
with solutions containing high concentrations of salts,
change of pH, and/or chelators that bind divalent cations.
Some cytosolic proteins have domains that bind to polar
head groups of lipids that transiently exist in a membrane.
The enzymes that create or degrade these lipids are subject
to signal-mediated regulation, providing a mechanism for
modulating affinity of a protein for a membrane surface.
O
E.g., pleckstrin
O H2C O C R2
homology (PH)
domains bind to R1 C O CH O

phosphorylated H2C O P O
derivatives of O- H
6
phosphatidylinositol. OH
1
OPO32-
H OH
2 5
Some PH domains OH H
PIP2 H
bind PIP2 (PI-4,5-P2). H
phosphatidylinositol- 3 4
H OPO32-
4,5-bisphosphate
 O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H
1 6
OH OH
phosphatidyl- H OH
inositol- 2
OPO32- H
5
3-phosphate H H
3 4
H OH

Other pleckstrin homology domains recognize and bind

phosphatidylinositol derivatives with Pi esterified at the
3' OH of inositol. E.g., PI-3-P, PI-3,4-P2, PI-3,4,5-P3.
 lipid
anchor O O C

H3C (CH2)14 C S CH2 CH cysteine

residue
membrane NH
palmitate

Some proteins bind to membranes via a covalently

attached lipid anchor, that inserts into the bilayer.
A protein may link to the cytosolic surface of the plasma
membrane via a covalently attached fatty acid (e.g.,
palmitate or myristate) or an isoprenoid group.
Palmitate is usually attached via an ester linkage to the
thiol of a cysteine residue.
A protein may be released from plasma membrane to
cytosol via depalmitoylation, hydrolysis of the ester link.
 CH3 CH3 CH3

H3C C CH CH2 CH2 C CH CH2 CH2 C CH CH2 S Protein

farnesyl residue linked to protein via cysteine S

An isoprenoid such as a farnesyl lipid

residue, is attached to some proteins anchor
via a thioether linkage to a cysteine thiol.
membrane
Glycosylphosphatidylinositols (GPI) are complex
glycolipids that attach some proteins to the outer surface
of the plasma membrane.
The linkage is similar to the following, although the
oligosaccharide composition may vary:
protein (C-term.) - phosphoethanolamine – mannose - mannose -
mannose - N-acetylglucosamine – inositol (of PI in membrane)

The protein is tethered some distance out from the

membrane surface by the long oligosaccharide chain.
GPI-linked proteins may be released from the outer
cell surface by phospholipases.
 peripheral
lipid
anchor

lipid bilayer

Membrane
integral Proteins

Integral proteins have domains that extend into the

hydrocarbon core of the membrane.
Often they span the bilayer.
Intramembrane domains have largely hydrophobic
surfaces, that interact with membrane lipids.
 membrane
Amphipathic
detergents are detergent polar
required for solubilization non-polar
solubilization of
integral proteins Protein with
from membranes. bound detergent

w Hydrophobic domains of detergents substitute for

lipids, coating hydrophobic surfaces of integral proteins.
w Polar domains of detergents interact with water.
If detergents are removed, purified integral proteins tend
to aggregate & come out of solution. Their hydrophobic
surfaces associate to minimize contact with water.
Lipid rafts:
w Sphingolipids in plasma membranes tend to separate
out from glycerophospholipids, & co-localize with
cholesterol in microdomains called lipid rafts.
w Lipid rafts are resistant to detergent solubilization,
which has facilitated their isolation & characterization.
w Differences in lipid shape may contribute to the
tendency for sphingolipids to separate out from
glycerophospholipids & associate with cholesterol.
• Sphingolipids usually lack double bonds in their
hydrocarbon chains.
• Glycerophospholipids often include at least one fatty
acid that is kinked, due to one or more double bonds.
w Lipid raft domains tend to be thicker than adjacent
membrane areas, in part because the saturated
hydrocarbon chains of sphingolipids are more extended.
w Proteins with fatty acyl or glycosylphosphatidylinositol
lipid anchors tend to associate preferentially with raft
domains.
E.g., proteins involved in cell signaling often assemble
in complexes at the cytosolic surface of the plasma
membrane in part by insertion of attached fatty acyl
groups into raft domains.
w Integral proteins may concentrate in raft domains via
interactions with raft lipids or with other raft proteins.
w Caveolae are invaginated lipid
raft domains of the plasma
membrane that have roles in cell caveolae
signaling and membrane cytosol
internalization.
w Caveolin is a protein associated with the cytosolic leaflet
of the plasma membrane in caveolae.
Caveolin interacts with cholesterol and self-associates
as oligomers that may contribute to deforming the
membrane to create the unique morphology of caveolae.
Integral protein structure
Atomic-resolution structures have been determined
for a small (but growing) number of integral membrane
proteins.
Integral proteins are difficult to crystallize for X-ray
analysis.
Because of their hydrophobic
transmembrane domains,
detergents must be present C
during crystallization.
membrane
A membrane-spanning a-helix is
the most common structural motif
found in integral proteins. N
 a-helix
R-groups in magenta

In an a-helix, amino acid R-groups protrude out from the

helically coiled polypeptide backbone.
The largely hydrophobic R-groups of a membrane-
spanning a-helix contact the hydrophobic membrane core,
while the more polar peptide backbone is buried.
Colors: C N O R-group (H atoms not shown).
alanine (Ala, A) isoleucine (Ile, I) leucine (Leu, L) valine (Val, V)
H H H H

H3N+ C COO- H3N+ C COO- H3N+ C COO- H3N+ C COO-

CH3 CH CH3 CH2 CH CH3

CH2 CH CH3 CH3

CH3 CH3
amino acids: non-polar aliphatic R-groups

Particular amino acids tend to occur at different

positions relative to the surface or interior of the bilayer
in transmembrane segments of integral proteins.
Residues with aliphatic side-chains (leucine, isoleucine,
alanine, valine) predominate in the middle of the bilayer.
 tryptophan tyrosine
H H

H2N C COO- H3N+ C COO-

CH2 CH2

Tyrosine and
tryptophan are HN
common near the
membrane surface. OH

It has been suggested that the polar character of the

tryptophan amide group and the tyrosine hydroxyl, along
with their hydrophobic ring structures, suit them for
localization at the polar/apolar interface.
 lysine arginine
H H

H3N+ C COO- H3N+ C COO-

CH2 CH2

CH2 CH2

CH2 CH2

CH2 NH
+
+ NH3 C NH2

NH2

Lysine & arginine are often at the lipid/water interface,

with the positively charged groups at the ends of their
aliphatic side chains extending toward the polar
membrane surface.
A 20-amino acid a-helix just spans a lipid bilayer.

Hydropathy plots are used to search for 20-amino

acid stretches of hydrophobic amino acids in the
primary sequence of a protein for which a crystal
structure is not available.
Putative hydrophobic transmembrane a-helices have
been identified this way in many membrane proteins.
Hydropathy plots alone are not conclusive.
Protein topology studies are used to test the
transmembrane distribution of protein domains
predicted by hydropathy plots.
 C

membrane

N C N

w If a hydropathy plot indicates one 20-amino acid

hydrophobic stretch (1 putative transmembrane a-helix),
topology studies are expected to confirm location of
N & C termini on opposite sides of membrane.
w If two transmembrane a-helices are predicted, N & C
termini should be on the same side. The segment
between the a-helices should be on the other side.
Transmembrane topology is tested with impermeant
probes, added on one side of a membrane. For example:
w Protease enzymes. Degradation a protein segment
indicates exposure to the aqueous phase on the side of
the membrane to which a protease is added.
w Monoclonal antibodies raised to peptides equivalent
to individual segments of the protein. Binding
indicates surface exposure of a protein segment on the
side to which the Ab is added.
Such studies have shown that all copies of a given type of
integral protein have the same orientation relative to the
membrane. Flip-flop of integral proteins does not occur.
 Simplified helical wheel diagram of four
a-helices lining the lumen of an ion channel.

A “helical
wheel” looks
down the axis
of an a-helix,
projecting side-
chains onto a Polar amino acid R-group
plane. Non-polar amino acid R-group

An a-helix lining a water-filled channel might have

polar amino acid R-groups facing the lumen, & non-polar
R-groups facing lipids or other hydrophobic a-helices.
Such mixed polarity would prevent detection by a
hydropathy plot.
 Porin b-barrel
While transmembrane a-
helices are the most common
structural motif for integral Porin Monomer
proteins, a family of bacterial
outer envelope channel
proteins called porins have
instead b barrel structures.
A b barrel is a b sheet rolled
up to form a cylindrical pore.
At right is shown one channel
of a trimeric porin complex. PDB 1AOS
 = polar R group, = non-polar R group

In a b-sheet, amino acid R-groups alternately point above

& below the sheet.
Much of porin primary structure consists of alternating
polar & non-polar amino acids.
• Polar residues face the aqueous lumen.
• Non-polar residues are in contact with membrane lipids.
Membrane Transport
Transporters are of two general classes:
carriers and channels.
These are exemplified by two ionophores (ion carriers
produced by microorganisms):
valinomycin (a carrier)
gramicidin (a channel).
 Valinomycin

H3C CH3
CH O O O CH3 O
H H
N CH C O C C N C C O CH C

H CH H CH
H3C CH3 H3C CH3 3
L-valine D-hydroxy- D-valine L-lactic
isovaleric acid acid

Valinomycin is a carrier for K+.

It is a circular molecule, made up of 3 repeats of
the sequence shown above.
Puckering of the ring,
Valinomycin
stabilized by H-bonds, allows
valinomycin to closely O
surround a single unhydrated O O
K+ ion. K
+

Six oxygen atoms of the O O

O
ionophore interact with the
bound K+, replacing O atoms Hydrophobic
of waters of hydration.
Valinomycin is highly selective for K+ relative to Na+.
The smaller Na+ ion cannot simultaneously interact with
all 6 oxygen atoms within valinomycin.
Thus it is energetically less favorable for Na+ to shed its
waters of hydration to form a complex with valinomycin.
 Valinomycin
O
O O
+
K
O O
O
Hydrophobic

Whereas the interior of the valinomycin-K+ complex is

polar, the surface of the complex is hydrophobic.
This allows valinomycin to enter the lipid core of the
bilayer, to solubilize K+ within this hydrophobic milieu.
Crystal structure (at Virtual Museum of Minerals & Molecules).
 Val-K+ Val-K+

K+ K+

Val Val

membrane

Valinomycin is a passive carrier for K+. It can bind or

release K+ when it encounters the membrane surface.
Valinomycin can catalyze net K+ transport because it can
translocate either in the complexed or uncomplexed state.
The direction of net flux depends on the electrochemical
K+ gradient.
Proteins that act as carriers are too large to move
across the membrane.
They are transmembrane proteins, with fixed topology.
An example is the GLUT1 glucose carrier, in plasma
membranes of various cells, including erythrocytes.
GLUT1 is a large integral protein, predicted via
hydropathy plots to include 12 transmembrane
a-helices.
 conformation conformation
change change

Carrier-mediated solute transport

Carrier proteins cycle between conformations in

which a solute binding site is accessible on one side of
the membrane or the other.
There may be an intermediate conformation in which a
bound substrate is inaccessible to either aqueous phase.
With carrier proteins, there is never an open channel
all the way through the membrane.
 conformation conformation
change change

Carrier-mediated solute transport

The transport rate mediated by carriers is faster than

in the absence of a catalyst, but slower than with
channels.
A carrier transports one or few solute molecules per
conformational cycle, whereas a single channel opening
event may allow flux of many thousands of ions.
Carriers exhibit Michaelis-Menten kinetics.
 Uniport Symport Antiport
Classes of A A B A
carrier
proteins
B

Uniport (facilitated diffusion) carriers mediate

transport of a single solute.
An example is the GLUT1 glucose carrier.
The ionophore valinomycin is also a uniport carrier.
 Uniport Symport An
Symport (cotransport) carriers
bind two dissimilar solutes A A B
(substrates) & transport them
together across a membrane.
Transport of the two solutes is
obligatorily coupled.
A gradient of one substrate, usually an ion, may drive uphill
(against the gradient) transport of a co-substrate.
It is sometimes referred to as secondary active transport.
E.g: w glucose-Na+ symport, in plasma membranes
of some epithelial cells
w bacterial lactose permease, a H+ symport carrier.
 Uniport Symport A

A A B
Lactose permease catalyzes uptake
of the disaccharide lactose into
E. coli bacteria, along with H+,
driven by a proton electrochemical
gradient.

It is the first carrier protein for which an atomic

resolution structure has been determined.
Lactose permease has been crystallized with
thiodigalactoside (TDG), an analog of lactose.
The substrate binding TDG
site is at the apex of an substrate
analog
aqueous cavity between
two domains, each
consisting of six trans-
membrane a-helices. Lactose Permease PDB 1PV7

In the conformation observed in this crystal structure,

the substrate analog is accessible only to what would be
the cytosolic side of the intact membrane.
Residues essential for H+ binding are are also near the
middle of the membrane.
 conformation conformation
change change

Carrier-mediated solute transport

As in simple models of
carrier transport based on
functional assays, the tilt of
transmembrane a-helices
TDG
is assumed to change, substrate
shifting access of lactose & analog
H+ binding sites to the other
side of the membrane during
the transport cycle. Lactose Permease PDB 1PV7
 Uniport Symport Antiport

Antiport (exchange diffusion)

A carriers
A B A
exchange one solute for another across a
membrane.
w Usually antiporters exhibit "ping pong"
kinetics. B
A substrate binds & is transported.
Then another substrate binds & is transported in the
other direction.
w Only exchange is catalyzed, not net transport.
The carrier protein cannot undergo the conformational
transition in the absence of bound substrate.
 mitochondrial
matrix

ATP 4-

ADP 3-

adenine nucleotide translocase

Example of an antiport carrier:

Adenine nucleotide translocase (ADP/ATP exchanger)
catalyzes 1:1 exchange of ADP for ATP across the inner
mitochondrial membrane.
 Active
Transport ADP + Pi

S1 S2

ATP
Side 1 Side 2

Active transport enzymes couple net solute movement

across a membrane to ATP hydrolysis.
An active transport pump may be a uniporter or
antiporter.
ATP-dependent ion pumps are grouped into classes, based
on transport mechanism, genetic & structural homology.
P-class ion pumps are a gene family exhibiting
sequence homology. They include:
w Na+,K+-ATPase, in plasma membranes of most
animal cells is an antiport pump.
It catalyzes ATP-dependent transport of Na+ out of a
cell in exchange for K+ entering.
w (H+, K+)-ATPase, involved in acid secretion in the
stomach is an antiport pump.
It catalyzes transport of H+ out of the gastric
parietal cell (toward the stomach lumen) in
exchange for K+ entering the cell.
P-class pumps (cont):
w Ca++-ATPases, in endoplasmic reticulum (ER) and
plasma membranes, catalyze ATP-dependent transport
of Ca++ away from the cytosol, into the ER lumen or
out of the cell.
Some evidence indicates that these pumps may be
antiporters, transporting protons in the opposite
direction.
Ca++-ATPase pumps function to keep cytosolic Ca++
low, allowing Ca++ to serve as a signal.
 O

Enzyme-C OH

ATP Pi

The reaction mechanism ADP H2O

O O
for a P-class ion pump
involves transient Enzyme- C O P O-
covalent modification
O-
of the enzyme. P-Class Pumps

At one stage of the reaction cycle, phosphate is transferred

from ATP to the carboxyl of a Glu or Asp side-chain,
forming a “high energy” anhydride linkage (~P).
At a later stage in the reaction cycle, the Pi is released by
hydrolysis.
 E~P-Ca++2 E~P-Ca++2
ADP
2Ca++
ATP E-Ca++2
The ER Ca++ pump
is called SERCA: 2Ca++
E
Sarco(Endo)plasmic
Reticulum Pi
ER
Ca++-ATPase. cytosol membrane lumen

In this diagram of SERCA reaction cycle,

conformational changes altering accessibility of
Ca++-binding sites to the cytosol or ER lumen are
depicted as positional changes.
Keep in mind that SERCA is a large protein that
maintains its transmembrane orientation.
Reaction cycle: E~P-Ca++2 E~P-Ca++2
ADP
1. 2 Ca++ bind tightly 2Ca++
from the cytosolic ATP E-Ca++2
side, stabilizing the
conformation that 2Ca++
E
allows ATP to react
with an active site Pi
ER
aspartate residue. cytosol membrane lumen

2. Phosphorylation of the active site aspartate induces a

conformational change that
• shifts accessibility of the 2 Ca++ binding sites from
one side of the membrane to the other, &
• lowers the affinity of the binding sites for Ca++.
 E~P-Ca++2 E~P-Ca++2
ADP
2Ca++
ATP E-Ca++2

2Ca++
E
Pi
ER
cytosol membrane lumen

3. Ca++ dissociates into the ER lumen.

4. Ca++ dissociation promotes
• hydrolysis of Pi from the enzyme Asp
• conformational change (recovery) that causes Ca++
binding sites to be accessible again from the cytosol.
 Asp351
cytosolic
This X-ray structure domain
of muscle SERCA
(Ca++-ATPase) shows
2 Ca++ ions (colored
magenta) bound between 2 Ca++ membrane
domain
transmembrane a-helices
in the membrane domain.
PDB 1EUL Muscle SERCA

Active site Asp351, which is transiently phosphorylated

during catalysis, is located in a cytosolic domain, far
from the Ca++ binding sites.
 ++ SERCA Conformational Cycle
Ca

enzyme phosphate
phosphorylation hydrolysis

This simplified cartoon represents the proposed

variation in accessibility & affinity of Ca++-binding sites
during the reaction cycle.
Only 2 transmembrane a-helices are represented, and the
cytosolic domain of SERCA is omitted.
 conformation
change

Ion
Channels closed open

Channels cycle between open & closed conformations.

When open, a channel provides a continuous pathway
through the bilayer, allowing flux of many ions.
Gramicidin is an example of a channel.
Gramicidin is an unusual peptide,
with alternating D & L amino acids.
In lipid bilayer membranes,
gramicidin dimerizes & folds as a
right-handed b-helix.
The dimer just spans the bilayer. Gramicidin dimer
(PDB file 1MAG)
Primary structure of gramicidin (A):
HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-
L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-
NHCH2CH2OH Note: The amino acids are all
hydrophobic; both peptide ends are modified (blocked).
The outer surface of the
gramicidin dimer, which interacts
with the core of the lipid bilayer,
is hydrophobic.
Ions pass through the more polar
lumen of the helix.
Ion flow through individual
gramicidin channels can be
observed if a small number of
gramicidin molecules is present in Gramicidin dimer
a lipid bilayer separating 2 (PDB file 1MAG)
compartments containing salt
solutions.
 Proposed mechanism of
gramicidin gating
Gating (opening &
closing) of a
gramicidin channel
is thought to
involve reversible
dimerization. open closed

An open channel forms when two gramicidin molecules

join end to end to span the membrane.
This model is consistent with the finding that at high
[gramicidin] overall transport rate depends on
[gramicidin]2.
 Channels that are proteins

Cellular channels usually consist of large protein

complexes with multiple transmembrane a-helices.
Their gating mechanisms must differ from that of
gramicidin.
Control of channel gating is a form of allosteric
regulation. Conformational changes associated with
channel opening may be regulated by:
w Voltage
w Binding of a ligand (a regulatory molecule)
w Membrane stretch (e.g., via link to cytoskeleton)
Signal Transduction
 serine (Ser) threonine (Thr)
H H

H3N+ C COO- H3N+ C COO-

CH2 CH OH

OH CH3

Many enzymes are regulated by covalent attachment

of phosphate, in ester linkage, to the side-chain
hydroxyl group of a particular amino acid residue
(serine, threonine, or tyrosine).
 Protein Kinase O

Protein OH + ATP Protein O P O- + ADP

O-
Pi H2O
Protein Phosphatase

w A protein kinase transfers the terminal phosphate of

ATP to a hydroxyl group on a protein.
w A protein phosphatase catalyzes removal of the Pi by
hydrolysis.
Phosphorylation may directly alter activity of an
enzyme, e.g., by promoting a conformational change.
Alternatively, altered activity may result from binding
another protein that specifically recognizes a
phosphorylated domain.
w E.g., 14-3-3 proteins bind to domains that include
phosphorylated Ser or Thr in the sequence
RXXX[pS/pT]XP, where X can be different amino
acids.
w Binding to 14-3-3 is a mechanism by which some
proteins (e.g., transcription factors) may be retained in
the cytosol, & prevented from entering the nucleus.
 Protein Kinase O

Protein OH + ATP Protein O P O- + ADP

O-
Pi H2O
Protein Phosphatase

Protein kinases and phosphatases are themselves

regulated by complex signal cascades. For example:
w Some protein kinases are activated by Ca++-
calmodulin.
w Protein Kinase A is activated by cyclic-AMP
(cAMP).
 Chi:
• Sistema endocrino (ormoni)
• Sistema nervoso (neurotrasmettitori)
• Sistema immunitario (citochine)

• retinoidi, eicosanoidi e fattori di

crescita Distanza:
• Effetto endocrino
• effetto paracrino
• effetto autocrino
affinità • Effetto intracrino
Classificazione:
• Composizione chimica
• Solubilità
• Localizzazione dei recettori
• Natura del segnale
 Classes of Hormones
• Steroid Hormones derived from cholesterol-
regulate metabolism, salt/water balances,
inflammation, sexual function
• Amino Acid Derived Hormones - epinephrine,
etc.- regulate smooth muscle, blood pressure,
cardiac rate, lipolysis, glycogenolysis
• Peptide Hormones - regulate many processes in
all tissues - including release of other hormones
77
 Types of Receptors
Three that we know of...
• 7-transmembrane segment receptors
– extracellular site for hormone (ligand)
– intracellular site for GTP-binding protein
• Single-transmembrane segment receptors
– extracellular site for hormone (ligand)
– intracellular catalytic domain - either a tyrosine
kinase or guanylyl cyclase
• Oligomeric ion channels
Recettori accoppiati alle proteine G (GPCR)

Recettori con attività tirosina chinasica (TRK)

Recettori con attività guanililciclasica

Recettori ionici controllati

Recettori di adesione cellulare

Recettori Notch

Recettori citoplasmatici e nucleari

Recettori accoppiati alle proteine G (GPCR):
(ATCH, TSH, catecolamine, LH, FSH)

Solitamente monomeri, ma alcuni sono presenti come

omo e eterodimeri
 7-TMS Receptors
Receptors that interact with G proteins
• Seven putative alpha-helical transmembrane
segments
• Extracellular domain interacts with hormone
• Intracellular domain interacts with G proteins
• Adrenergic receptors are typical
• Note desensitization by phosphorylation, either by
bARK or by protein kinase A
 emivita 5 min
fegato
adipociti

86
 Second Messengers
Many and there may be more!
• The hormone is the "first messenger"
• The second messenger - Ca2+, cAMP or other - is
released when the hormone binds to its
(extracellular) receptor
• The second messenger then activates (or inhibits)
processes in the cytoplasm or nucleus
• Degradation and/or clearance of the second
messenger is also (obviously) important
 cAMP and Glycogen
Phosphorylase
Earl Sutherland discovers the first second messenger
• In the early 1960s, Earl Sutherland showed that the
stimulation of glycogen phosphorylase by
epinephrine involved cyclic adenosine-3',5'-
monophosphate
• He called cAMP a "second messenger"
• cAMP is synthesized by adenylyl cyclase and
degraded by phosphodiesterase
Adenylate Cyclase (Adenylyl
cAMP NH2
Cyclase) catalyzes:
ATP à cAMP + PPi N
N
Binding of certain hormones
(e.g., epinephrine) to the outer N N
surface of a cell activates
H2 O
Adenylate Cyclase to form 5' C 4'
H H 1'
cAMP within the cell. O
H 3' 2' H
Cyclic AMP is thus considered P O OH
O
to be a second messenger. O-
 G Proteins
Many new developments in this area
• Two kinds: "heterotrimeric G proteins" and
"small G proteins"
• X-ray diffraction structures for several of
these are only recently available
• Structures shed new light on possible
functions
 Heterotrimeric G Proteins
A model for their activity
• Binding of hormone, etc., to receptor protein in the
membrane triggers dissociation of GDP and
binding of GTP to a-subunit of G protein
• Ga-GTP complex dissociates from Gbg and
migrates to effector sites, activating or inhibiting
• But it is now clear that Gbg also functions as a
signalling device
Phosphodiesterase enzymes NH2
cAMP
catalyze:
cAMP + H2O à AMP N
N

The phosphodiesterase that N

N
cleaves cAMP is activated by
phosphorylation catalyzed by H2 O
5' C 4'
Protein Kinase A. H H 1'
O
H 3' 2' H
Thus cAMP stimulates its own P O OH
degradation, leading to rapid O
O-
turnoff of a cAMP signal.
Protein Kinase A (cAMP-Dependent Protein Kinase)
transfers Pi from ATP to OH of a Ser or Thr in a
particular 5-amino acid sequence.
Protein Kinase A in the resting state is a complex of:
• 2 catalytic subunits (C)
• 2 regulatory subunits (R).
R2C2
R2C2
Each regulatory subunit (R) of Protein Kinase A
contains a pseudosubstrate sequence, like the
substrate domain of a target protein but with Ala
substituting for the Ser/Thr.
The pseudosubstrate domain of (R), which lacks a
hydroxyl that can be phosphorylated, binds to the
active site of (C), blocking its activity.
R2C2 + 4 cAMP à R2cAMP4 + 2 C
When each (R) binds 2 cAMP, a conformational
change causes (R) to release (C).
The catalytic subunits can then catalyze
phosphorylation of Ser or Thr on target proteins.

PKIs, Protein Kinase Inhibitors, modulate activity of

the catalytic subunits (C).
G Protein Signal Cascade
Most signal molecules targeted to a cell bind at the cell
surface to receptors embedded in the plasma membrane.
Only signal molecules able to cross
the plasma membrane (e.g., steroid
hormones) interact with intracellular
receptors.
A large family of cell surface
receptors have a common structural
motif, 7 transmembrane a-helices.
Rhodopsin was the 1st member of
this family to have its 7-helix
structure confirmed by X-ray
crystallography. Rhodopsin PDB 1F88
w Rhodopsin is unique in that it
senses light.
w Most 7-helix receptors have
domains facing the extracellular
side of the plasma membrane that
recognize & bind particular
signal molecules (ligands).
Rhodopsin PDB 1F88
The signal is passed from a 7-helix
receptor to an intracellular G-protein.
w Seven-helix receptors are thus called GPCR, or
G-Protein-Coupled Receptors.
w Approx. 800 different GPCRs are encoded in the
human genome.
G-protein-Coupled Receptors may dimerize or form
oligomeric complexes within the membrane.
Ligand binding may promote oligomerization, which
may in turn affect activity of the receptor.
Various GPCR-interacting proteins (GIPs) modulate
receptor function. Effects of GIPs may include:
w altered ligand affinity
w receptor dimerization or oligomerization
w control of receptor localization, including transfer to
or removal from the plasma membrane
w promoting close association with other signal proteins
w G-proteins are heterotrimeric, with 3 subunits a, b, g.
w A G-protein that activates cyclic-AMP formation
within a cell is called a stimulatory G-protein,
designated Gs with alpha subunit Gsa.
w Gs is activated, e.g., by receptors for the hormones
epinephrine and glucagon.
The b-adrenergic receptor is the GPCR for
epinephrine.
 hormone
signal

outside

GPCR plasma
membrane
The a subunit of
a G-protein (Ga) a g g + a cytosol
binds GTP, & GDP b b GTP
AC
can hydrolyze it
to GDP + Pi. GTP GDP ATP cAMP + PP i

a & g subunits have covalently attached lipid anchors that

bind a G-protein to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with
cytosolic domains forming the catalytic site.
 hormone
signal

outside

GPCR plasma
The complex membrane

of b & g
a g g + a cytosol
subunits Gb,g AC
GDP b b GTP
inhibits Ga.
GTP GDP ATP cAMP + PP i

The sequence of events by which a hormone activates

cAMP signaling:
1. Initially Ga has bound GDP, and a, b, & g subunits
are complexed together.
 hormone
signal

outside

GPCR plasma
membrane

a g g + a cytosol
AC
GDP b b GTP

GTP GDP ATP cAMP + PP i

2. Hormone binding to a 7-helix receptor (GPCR)

causes a conformational change in the receptor that is
transmitted to the G protein.
The nucleotide-binding site on Ga becomes more
accessible to the cytosol, where [GTP] > [GDP].
Ga releases GDP & binds GTP (GDP-GTP exchange).
 hormone
signal

outside

GPCR plasma
membrane

a g g + a cytosol
AC
GDP b b GTP

GTP GDP ATP cAMP + PP i

3. Substitution of GTP for GDP causes another

conformational change in Ga.
Ga-GTP dissociates from the inhibitory bg complex &
can now bind to and activate Adenylate Cyclase.
 hormone
signal

outside

GPCR plasma
membrane

a g g + a cytosol
AC
GDP b b GTP

GTP GDP ATP cAMP + PP i

4. Adenylate Cyclase, activated by the stimulatory

Ga-GTP, catalyzes synthesis of cAMP.
5. Protein Kinase A (cAMP Dependent Protein Kinase)
catalyzes phosphorylation of various cellular proteins,
altering their activity.
Turn off of the signal:
1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Ga causes it to rebind to the
inhibitory bg complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
cAMP à AMP.
Turn off of the signal (cont.):
3. Receptor desensitization occurs. This process varies
with the hormone.
w Some receptors are phosphorylated via specific
receptor kinases.
w The phosphorylated receptor may then bind to a
protein b-arrestin, that promotes removal of the
receptor from the membrane by clathrin-mediated
endocytosis.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to proteins
via Protein Kinase A.
Signal amplification is an important feature of signal
cascades:

w One hormone molecule can lead to formation of

many cAMP molecules.

w Each catalytic subunit of Protein Kinase A

catalyzes phosphorylation of many proteins during
the life-time of the cAMP.
w The stimulatory Gsa, when it binds GTP,
activates Adenylate cyclase.
w An inhibitory Gia, when it binds GTP, inhibits
Adenylate cyclase.
Different effectors & their receptors induce Gia to
exchange GDP for GTP than those that activate Gsa.
In some cells, the complex of Gb,g that is released
when Ga binds GTP is itself an effector that binds to
and activates other proteins.
w Cholera toxin catalyzes covalent modification of Gsa.
• ADP-ribose is transferred from NAD+ to an arginine
residue at the GTPase active site of Gsa.
• ADP-ribosylation prevents GTP hydrolysis by Gsa .
• The stimulatory G-protein is permanently activated.
w Pertussis toxin (whooping cough disease) catalyzes ADP-
ribosylation at a cysteine residue of the inhibitory Gia,
making it incapable of exchanging GDP for GTP.
• The inhibitory pathway is blocked.
w ADP-ribosylation is a general mechanism by which
activity of many proteins is regulated, in eukaryotes
(including mammals) as well as in prokaryotes.
 ADP H O
protein
(CH2)3
ribosylation C
NH2
NH
O +
- N C NH2+
O P O CH2 O
H H O
- NH
H H O P O CH2 O
OH OH
H H
O NH2 H H
N OH OH
N O NH2
N
O P O CH2 N
- N N
O
O H H -
O P O CH2 N N
+ H H O
NAD OH OH H H
O
(nicotinamide H H
protein OH OH
adenine H O
dinucleotide) (CH2)3 ADP-ribosylated
C
NH NH2 protein
Arg +
C NH2+ N
residue nicotinamide
H
NH2
Structure of G proteins: PDB 1GIA

The nucleotide binding site

in Ga consists of loops that
extend out from the edge of
a 6-stranded b-sheet.
Three switch domains have
been identified, that change GTPgS
position when GTP
substitutes for GDP on Ga. Inhibitory Ga
These domains include residues adjacent to the terminal
phosphate of GTP and/or the Mg++ associated with the
two terminal phosphates.
 O

GTP hydrolysis N
NH
H

H O O O O N N NH2
-
O P O P O P O CH2
O
O- O- O- H H
H H
OH OH

GTP hydrolysis occurs by nucleophilic attack of a

water molecule on the terminal phosphate of GTP.
Switch domain II of Ga includes a conserved
glutamine residue that helps to position the attacking
water molecule adjacent to GTP at the active site.
 PDB 1GP2

PDB 1GP2

Gb - side view of b-propeller

Gb – face view of b-propeller

The b subunit of the heterotrimeric G Protein has a

b-propeller structure, formed from multiple repeats of a
sequence called the WD-repeat.
The b-propeller provides a stable structural support for
residues that bind Ga.
The family of heterotrimeric G proteins includes also:
w transducin, involved in sensing of light in the retina.
w G-proteins involved in odorant sensing in olfactory
neurons.

There is a larger family of small GTP-binding switch

proteins, related to Ga.
 Signalling Roles for G(bg)
A partial list
• Potassium channel proteins
• Phospholipase A2
• Adenylyl cyclase
• Phospholipase C
• Calcium channels
• Receptor kinases
 Stimulatory and Inhibitory G
G proteins may either stimulate or inhibit an effector.
• In the case of adenylyl cyclase, the stimulatory G
protein is known as Gs and the inhibitory G protein is
known as Gi
• Gi may act either by the Gia subunit binding to AC or
by the Gibg complex complexing all the Gia and
preventing it from binding to AC
• Read about the actions of cholera toxin and pertussis
toxin
Somatostatina
cellule d isole pancreatiche
 Phospholipases Release Second
Messengers
• Inositol phospholipids yield IP3 and DAG
• PLCb is activated by 7-TMS receptors and G
proteins
• PLCg is activated by receptor tyrosine kinases
(via phosphorylation)
• Note PI metabolic pathways and the role of
lithium
 Other Lipids as Messengers
Recent findings - lots more to come
• More recently than for PI, other
phospholipids have been found to produce
second messengers!
• PC can produce C20s, DAG and/or PA
• Sphingomyelin and glycosphingolipids also
produce signals
• Ceramide (from SM) is a trigger of apoptosis
- programmed cell death
Phosphatidylinositol Signal Cascades
O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H
1 6
OH OH
H OH
2 H 5
OH
phosphatidyl- H H
3 4
inositol H OH

Some hormones activate a signal cascade based on the

membrane lipid phosphatidylinositol.
 O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H
1 6
OH OPO32-
H OH
2 OH H 5

H H
PIP2 3 4
phosphatidylinositol- H OPO32-
4,5-bisphosphate

Kinases sequentially catalyze transfer of Pi from ATP to

OH groups at positions 5 & 4 of the inositol ring, to yield
phosphatidylinositol-4,5-bisphosphate (PIP2).
PIP2 is cleaved by the enzyme Phospholipase C.
 O
Different isoforms O H2C O C R2
of Phospholipase C R1 C O CH O
have different
H2C O P O
regulatory domains,
cleavage by O- H
& thus respond to 1 6
Phospholipase C OH OPO32-
different signals. H OH
5
2 OH H
A G-protein, Gq H H
PIP2 3
activates one form phosphatidylinositol- H
4
OPO32-
of Phospholipase C. 4,5-bisphosphate

When a particular GPCR (receptor) is activated, GTP

exchanges for GDP. Gqa-GTP activates Phospholipase C.
Ca++, which is required for activity of Phospholipase C,
interacts with (-) charged residues & with Pi moieties of
the phosphorylated inositol at the active site.
 OPO32- H
1 6
OH OPO32- O
H OH
2 OH H 5 O H2C O C R2
H H R1 C O CH
3 4
H OPO32- H2C OH
IP3
inositol-1,4,5-trisphosphate diacylglycerol

Cleavage of PIP2, catalyzed by Phospholipase C, yields 2

second messengers:
w inositol-1,4,5-trisphosphate (IP3)
w diacylglycerol (DG).
Diacylglycerol, with Ca++, activates Protein Kinase C,
which catalyzes phosphorylation of several cellular
proteins, altering their activity.
 Ca++ calmodulin

IP3 Ca++-release channel

++
endoplasmic
Ca reticulum

Ca++-ATPase
ATP ++ ADP + Pi
Ca

IP3 activates Ca++-release channels in ER membranes.

Ca++ stored in the ER is released to the cytosol, where it
may bind calmodulin, or help activate Protein Kinase C.
Signal turn-off includes removal of Ca++ from the
cytosol via Ca++-ATPase pumps, & degradation of IP3.
 OPO32- H OH H

OH OPO32- OH OH
H OH H OH
OH H OH H + 3 Pi
(3 steps)
H H H H

H OPO32- H OH
IP3 inositol

Sequential dephosphorylation of IP3 by enzyme-catalyzed

hydrolysis yields inositol, a substrate for synthesis of PI.
IP3 may instead be phosphorylated via specific kinases,
to IP4, IP5 or IP6. Some of these have signal roles.
E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells
stimulates Ca++ entry, perhaps by activating plasma
membrane Ca++ channels.
 O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H
1 6
OH OH
phosphatidyl- H OH
inositol- 2
OPO32- H
5
3-phosphate H H
3 4
H OH

The kinases that convert PI (phosphatidylinositol) to

PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions
4 & 5 of the inositol ring.
PI 3-Kinases instead catalyze phosphorylation of
phosphatidylinositol at the 3 position of the inositol ring.
Signal protein complexes:
Signal cascades are often mediated by large "solid state"
assemblies that may include receptors, effectors, and
regulatory proteins, linked together in part by interactions
with specialized scaffold proteins.
Scaffold proteins often interact also with membrane
constituents, elements of the cytoskeleton, and adaptors
mediating recruitment into clathrin-coated vesicles.
They improve efficiency of signal transfer, facilitate
interactions among different signal pathways, and control
localization of signal proteins within a cell.
Signal complexes are often associated with lipid raft
domains of the plasma membrane.
Scaffold proteins as well as signal proteins may be
recruited from the cytosol to such membrane domains
in part by
w insertion of lipid anchors
w interaction of pleckstrin homology or other lipid-
binding domains with head-groups of transiently
formed phosphatidylinositol derivatives, such as
PIP2 or PI-3-P.
AKAPs (A-Kinase Anchoring Proteins) are scaffold
proteins with multiple domains that bind to
w regulatory subunits of Protein Kinase A
w phosphorylated derivatives of phosphatidylinositol
w various other signal proteins, such as:
• G-protein-coupled receptors (GPCRs)
• Other kinases such as Protein Kinase C
• Protein phosphatases
• Phosphodiesterases
AKAPs localize hormone-initiated signal cascades within
a cell, and coordinate activation of protein kinases as
well as rapid turn-off of such signals.
 Ca2+ as a Second Messenger
Several sources of Ca2+ in cells!
• [Ca2+] in cells is normally very low: < 1µM
• Calcium can enter cell from outside or from ER and
calciosomes
• CICR - Calcium-Induced Calcium Release - is very,
very similar to what happens at the foot structure in
muscle cells!
• IP3 (made by action of phospholipase C) is the trigger
 Calcium Oscillations!
M. Berridge's model of Ca2+ signals
• Ca2+ was once thought to merely rise in cells to
signal and drop when the signal was over
• Berridge's work demonstrates that Ca2+ levels
oscillate in cells!
• The purpose may be to protect cell components
that are sensitive to high calcium, or perhaps to
create waves of Ca2+ in the cell
 Ca2+-Binding Proteins
Mediators of Ca2+effects in cells
• Many cellular proteins modulate Ca2+ effects
• 3 main types: protein kinase Cs, Ca2+-
modulated proteins and annexins
• Kretsinger characterized the structure of
parvalbumin, prototype of Ca2+-modulated
proteins
• "EF hand" proteins bind BAA helices
 Transduction of two second
messenger signals
PKC is activated by DAG and Ca2+
• Most PKC isozymes have several domains, including
ATP-binding domain, substrate-binding domain, Ca-
binding domain and a phorbol ester-binding domain
• Phorbol esters are apparent analogues of DAG
• Cellular phosphatases dephosphorylate target
proteins
 emivita 5 min
fegato
adipociti

146
 Single TMS Receptors
Three main classes
• Extracellular domain to interact with hormone
• Single transmembrane segment
• Intracellular domain with enzyme activity
• Activity is usually tyrosine kinase or guanylyl
cyclase
• Each of these has a "nonreceptor" counterpart
• src gene kinase - pp60v-src was first known
• Two posttranslational modifications
 Receptor Tyrosine Kinases
Membrane-associated allosteric enzymes
• How do single-TMS receptors transmit the
signal from outside to inside??
• Oligomeric association is the key!
• Extracellular ligand binding
fegato, muscolo e tessuto adiposo

insulinR

Il legame con il ligando attiva la NGF (nerve GF)

subunità citosolica che fosforila 3 PDGF (platelet derivated GF)
Tyr dell’altra subunità (a vicenda) VEGF(vascular endothelial GF)
 emivita 7-15 min

151
 Evento cruciale IRS-P
MAPKKK

G protein monomerica
MAPKK

Cascata
Ras-MAPK
comune
anche a
Proteina regolatoria non PDGF e EGF
enzimatica

Mitosi
DivIsione cell

fattore di scambio
152
Small GTP-binding proteins include (roles indicated):
w initiation & elongation factors (protein synthesis).
w Ras (growth factor signal cascades).
w Rab (vesicle targeting and fusion).
w ARF (forming vesicle coatomer coats).
w Ran (transport of proteins into & out of the nucleus).
w Rho (regulation of actin cytoskeleton)

All GTP-binding proteins differ in conformation

depending on whether GDP or GTP is present at their
nucleotide binding site.
Generally, GTP binding induces the active state.
 protein-GTP (active)
Most GTP-binding
proteins depend on GDP
helper proteins: GEF GAP
GAPs, GTPase Activating GTP Pi
Proteins, promote GTP
hydrolysis. protein-GDP (inactive)

A GAP may provide an essential active site residue,

while promoting the correct positioning of the glutamine
residue of the switch II domain.
Frequently a (+) charged arginine residue of a GAP
inserts into the active site and helps to stabilize the
transition state by interacting with (-) charged O atoms
of the terminal phosphate of GTP during hydrolysis.
 protein-GTP (active)

GDP

GEF GAP

GTP Pi

protein-GDP (inactive)

w Ga of a heterotrimeric G protein has innate capability

for GTP hydrolysis.
It has the essential arginine residue normally provided
by a GAP for small GTP-binding proteins.
w However, RGS proteins, which are negative
regulators of G protein signaling, stimulate GTP
hydrolysis by Ga.
 protein-GTP (active)

GDP

GEF GAP
GEFs, Guanine Nucleotide
GTP Pi
Exchange Factors, promote
GDP/GTP exchange. protein-GDP (inactive)

w An activated receptor (GPCR) normally serves as

GEF for a heterotrimeric G-protein.
w Alternatively, AGS (Activator of G-protein Signaling)
proteins may activate some heterotrimeric G-proteins,
independent of a receptor.
Some AGS proteins have GEF activity.
 The ras Gene and p21ras

An oncogene and its product

• a gene first found in rat sarcoma virus
• Normal cellular ras protein activates cellular
processes when GTP is bound and is inactive when
GTP has been hydrolyzed to GDP
• Mutant (oncogenic) forms of ras have severely
impaired GTPase activity, so remain active for long
periods, stimulating excessive growth and
metabolic activity - causing tumors to form
158
 O

O H2C O C R2
PI-3K
R1 C O CH O

H2C O P O

O- H
PI-3-P, PI-3,4-P2, 1 6
OH OH
PI-3,4,5-P3, and phosphatidyl- H OH
inositol- 2 2- 5
PI-4,5-P2 have 3-phosphate
OPO3 H
H H
signaling roles. 3 4
H OH

Head-groups of these transiently formed lipids are ligands

for particular pleckstrin homology (PH) & FYVE protein
domains that bind proteins to membrane surfaces.
PI-3Ks : tutte kinasi citosoliche (due tipi):
• Tipo A. attivate da ligandi a recettori TyrK
• Tipo B: attivate da ligandi GPCR
Protein Kinase B (also called Akt) becomes activated when it is
recruited from the cytosol to the plasma membrane surface by
binding to products of PI-3 Kinase, e.g., PI-3,4,5-P3.
w Other kinases at the cytosolic surface of the plasma membrane
then catalyze phosphorylation of Protein Kinase B, activating it.
w Activated Protein Kinase B catalyzes phosphorylation of Ser or
Thr residues of many proteins, with diverse effects on
metabolism, cell growth, and apoptosis.
w Downstream metabolic effects of Protein Kinase B include
stimulation of glycogen synthesis, stimulation of glycolysis, and
inhibition of gluconeogenesis.
w Akt fosforila la proteina antiapoptotica BAD attivandola
 OPO32- H OH H

OH OPO32- OH OH
H OH H OH
OH H OH H + 3 Pi
(3 steps)
H H H H

H OPO32- H OH
IP3 inositol

Sequential dephosphorylation of IP3 by enzyme-catalyzed

hydrolysis yields inositol, a substrate for synthesis of PI.

PTEN: fosfatasi PI-3,4,5 in PI-4,5

 emivita 7-15 min

GLUT-1: eritrociti
GLUT-2: epatociti
GLUT-3: cellule encefalo
GLUT-4: cellule muscolari/cardiache
162
w Lipid raft domains tend to be thicker than adjacent
membrane areas, in part because the saturated
hydrocarbon chains of sphingolipids are more extended.
w Proteins with fatty acyl or glycosylphosphatidylinositol
lipid anchors tend to associate preferentially with raft
domains.
E.g., proteins involved in cell signaling often assemble
in complexes at the cytosolic surface of the plasma
membrane in part by insertion of attached fatty acyl
groups into raft domains.
w Integral proteins may concentrate in raft domains via
interactions with raft lipids or with other raft proteins.
w Caveolae are invaginated lipid raft domains of
the plasma membrane that have roles in cell
signaling and membrane internalization.
caveolae
cytosol
w Adipociti: INS-R forma un complesso con
caveolina-1 (regione ricca lipid raft) che
favorisce il reclutamento di GLUT-4 in
membrana
 Recettori citochinici
(GH, prolattina, EPO)

Sprovvisti di attività tirosina chinasica intrinseca

attivano altri enzimi (solubili) come ad esempio la
Janus Kinase 2 (JAK2) (tirosina chinasi citosolica) che viene attivata dal
legame di GH al recettore dimero.

L’attivazione di JAK2 determina la sua autofosforilazione e la successiva

fosforilazione del recettore che acquisisce capacità di interagire con STAT
che migrerà nel nucleo per legarsi a specifiche regioni di DNA con
aumento della trascrizione di alcuni geni come quello per IGF-1
166
 Recettori TGFb
(TGFb, inibina, attivina, ormone antimulleriano e BMP)

attività Ser/Thr chinasica intrinseca

Tipo I
Tipo II (sempre attivo e responsabile della fosforilazione del tipo I)

Effettori: proteine della famiglia SMAD che fosforilate traslocano nel

nucleo
 Recettori…
• Recettori ad attività tirosina fosfatasica:
attività del dominio citosolico in seguito a
legame con il ligando extracellulare
• Plexine: R transmembrana interagiscono
con semaforine (proteine) e hanno come
effettori G protein monomeriche (RAC,
RAS..)
• Integrine: eterodimeri (ab) giunzioni
adesive tra cellule (adesione focale)
 Protein-Tyrosine Phosphatases
The enzymes that dephosphorylate Tyr
• Some PTPases are integral membrane proteins
• But there are also lots of soluble PTPases
• Cytoplasmic PTPases have N-term. catalytic
domains and C-terminal regulatory domains
• Membrane PTPases all have cytoplasmic catalytic
domain, single transmembrane segment and an
extracellular recognition site
 Recettori Notch
• Recettori coinvolti nella regolazione della
proliferazione e apoptosi
• il recettore nella porzione extracellulare lega il
ligando presente sulla superficie di un’altra
cellula, favorendo l’attacco di proteasi di
membrana ADAM 10 (porzione extracellulare) e
g-secretasi per la propria porzione intracellulare
(dominio TAD e PEST) che migra nel nucleo
• Cellule che condividono potenziale differenziativo
 The Polypeptide Hormones
Common features of synthesis
• All secreted polypeptide hormones are synthesized
with a signal sequence (which directs them to
secretory granules, then out)
• Usually synthesized as inactive preprohormones
("pre-pro" implies at least two precessing steps)
• Proteolytic processing produces the prohormone
and the hormone
 Proteolytic Processing
A mostly common pathway
• Proteolytic cleavage of the hydrophobic N-
terminal signal peptide sequence
• Proteolytic cleavage at a site defined by pairs
of basic amino acid residues
• Proteolytic cleavage at sites designated by
single Arg residues
• Post-translational modification: C-terminal
amidation, N-terminal acetylation,
phosphorylation, glycosylation
 Gastrin as an Example
Heptadecapeptide secreted by the antral mucosa of
stomach
• Gastrin stimulates acid secretion in stomach
• Product of preprogastrin - 101-104 residues
• Signal peptide cleavage leaves progastrin - 80-83
residues
• Cleavage at Lys and Arg (basic) residues and C-
terminal amidation leaves gastrin
• N-terminal residue of gastrin is pyroglutamate
• C-terminal amidation involves destruction of Gly
 Protein Modules in Signal
Transduction
• Signal transduction in cell occurs via
protein-protein and protein-lipid
interactions based on protein modules
• Most signaling proteins consist of two or
more modules
• This permits assembly of functional
signaling complexes
 Localization of Signaling
Proteins
• Adaptor proteins provide docking sites for
signaling modules at the membrane
• Typical case: IRS-1 (Insulin Receptor
Substrate-1)
– N-terminal PH domain
– PTB domain
– 18 potential tyrosine phosphorylation sites
– PH and PTB direct IRS-1 to receptor tyrosine
kinase - signaling events follow!
 Signaling Pathways from
Membrane to the Nucleus
• The complete path from membrane to
nucleus is understood for a few cases
• Signaling pathways are redundant
• Signaling pathways converge and diverge
• This is possible with several signaling
modules on a signaling protein
 Module Interactions Rule!
• The interplay of multiple modules on many
signaling proteins permits a dazzling array
of signaling interactions
• We can barely conceive of the probable
extent of this complexity
• For example, it is estimated that there are
more than 1000 protein kinases in the
typical animal cell - all signals!
The transcription factor NF-κB is a heterodimeric complex
typically composed of two subunits termed p50 and p65

1
Recettori accoppiati alle proteine G (GPCR)

Recettori con attività tirosina chinasica (TRK)

Recettori con attività guanililciclasica

Recettori ionici controllati

Recettori di adesione cellulare

Recettori Notch

Recettori citoplasmatici e nucleari

 Guanylyl Cyclases
Soluble or Membrane-Bound
• Membrane-bound GCs are the other group of
single-transmembrane-segment receptors
(besides RTKs)
• Peptide hormones activate the membrane-
forms
• Note speract and resact, from mammalian ova
• Activation may involve oligomerization of
receptors, as for RTKs
Membrane-Bound GCs
cGMP attiva PKG espressa in: Vasodilatazione (ANP)

Meccanismo simile anche (BNP)

• cellule muscolari lisce
• piastrine
• cellule di Purkinje (cervello cervelletto)
• intestino
• rene

aumento volume sangue

(cellule atrio cardiaco)

peptide natriuretico atriale (ANP o atriopeptina)
Riduzione volemia

Rene (GC) di membrana

(tubuli collettori) Maggiore escrezione di Na+ richiamo di H2O
 GC sempre attiva

Elevate [cGMP]

Canali del Na+ sempre aperti

membrana ipopolarizzata
fotone

rodopsina (bastoncelli)
iodopsina (coni)
impulso nervoso
attivazione fosfodiesterasi

membrana iperpolarizzata chiusura dei canali ridotta [cGMP]

 Soluble Guanylyl Cyclases
Receptors for Nitric Oxide
• NO is a reactive, free-radical that acts either as a
neurotransmitter or as a second messenger
• NO relaxes vascular smooth muscle (and is thus
involved in stimulation of penile erection)
• NO also stimulates macrophages to kill tumor cells and
bacteria
• NO binds to heme of GC, stimulating GC activity 50-
fold
 NO diffonde rapidamente, emivita 1-5s,
Trasportato da Hb che lo rende più
stabile

NO sintasi (NOS) Ca2+ dipendente (eNOS)

Alcune citochine stimolano la iNOS (inducibile, no Ca2+)

Viagra e
Glucocorticoidi possono inibire iNOS e cGMP cGMP PDE
Recettori accoppiati alle proteine G (GPCR)

Recettori con attività tirosina chinasica (TRK)

Recettori con attività guanililciclasica

Recettori ionici controllati

Recettori di adesione cellulare

Recettori Notch

Recettori citoplasmatici e nucleari

 Steroid Hormones
Glucocorticoids, mineralocorticoids, vitamin D and
the sex hormones
• May either act at nucleus or at plasma membrane
• Steroids are hydrophobic and cannot diffuse freely
to nucleus
• Receptor proteins carry steroids to the nucleus
• Steroid receptor proteins are all apparently
members of a gene superfamily and have evolved
from a common ancestral precursor
 Recettori intracellulari:
• citosolici (corticosteroidi)
• nucleari (sessuali, tiroidei, vit D)

HSP heat shock protein

• Famiglia dei recettori steroidei

• Famiglia dei recettori tiroidei

12
 Extracellular Effects
of steroid hormones
• Two lines of evidence: action of steroids on calcium
channels and other membrane proteins and the speed
of certain steroid hormone effects
• Example: testosterone rapidly stimulates transport of
glucose, calcium and amino acids into rat kidney
cells
• Several demonstrations now of tight binding of
steroid probes to GABA receptor and other proteins
 Steroid Receptor Proteins
• Hydrophobic domain near C-terminus that interacts
with steroid itself
• Central, hydrophilic domain that binds to DNA
• Central DNA-binding domains are homologous to
one another, with 9 conserved Cys residues
• Three pairs of Cys residues are in Cys-X-X-Cys
sequences - as in Zinc-finger domains
• Steroid-receptor complex may bind to DNA or to
transcription factors
• Thyroid hormone receptor proteins are similar
Recettori tiroidei sono simili a quelli steroidei, ma non hanno c’è una
regione per il legame con HSP

A questa sottofamiglia appartengono:

• R. orm tiroidei
• R 1,25 Diidrossicolecalciferolo
• R. acido retinoico
• R. PPAR
ormoni
Hormones can be classified according to:
• chemical composition
• solubility properties
• location of receptors
• the nature of the signal used to mediate
hormonal action within the cell.
 The Polypeptide Hormones
Common features of synthesis
• All secreted polypeptide hormones are synthesized
with a signal sequence (which directs them to
secretory granules, then out)
• Usually synthesized as inactive preprohormones
("pre-pro" implies at least two precessing steps)
• Proteolytic processing produces the prohormone
and the hormone
 Proteolytic Processing
A mostly common pathway
• Proteolytic cleavage of the hydrophobic N-
terminal signal peptide sequence
• Proteolytic cleavage at a site defined by pairs
of basic amino acid residues
• Proteolytic cleavage at sites designated by
single Arg residues
• Post-translational modification: C-terminal
amidation, N-terminal acetylation,
phosphorylation, glycosylation
 Valenza
diagnostica
per diabetici
INS-dipendenti

65-80% tot cellule

24 AA

Proormone convertasi

51 AA in tutte le specie animali

 (circa 1 unità/kg pc)
Secrezione bifasica:
• I fase immediata (1-10 min) INS immagazzinata
• II fase più lunga INS di neosintesi

Glu entra in b-cells (mediante GLUT-2) e diventa Glu6P (sensore)

[ATP] Chiusura dei canali K+

depolarizzazione membrana e apertura Ca-channels

Legame con CaM e attivazione PKC Secrezione INS

Emivita 7-15 minuti

Sintesi INS
Inattivazione enzimi proteolitici lisosomiali

Fegato: glutatione-insulina trans-idrogenasi riduce i ponti S-S (utilizzando 4 GSH)

 Evento cruciale IRS-P
MAPKKK

G protein monomerica
MAPKK

Cascata
Ras-MAPK
comune
anche a
Proteina regolatoria non PDGF e EGF
enzimatica

Mitosi
DivIsione cell

fattore di scambio
32
Insulin

Insulin is a peptide hormone that functions in lowering blood glucose levels. Insulin has several activities that
accomplish this goal, summarized below:
1. Insulin inhibits transcription of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is a
key enzyme in gluconeogenesis and transcription is the primary means of regulating it. By inhibiting
PEPCK transcription, insulin can depress glucose production tremendously. (Conversely, the hormone
glucagon, which increases blood glucose levels, stimulates PEPCK transcription.)
2. Insulin stimulates translocation of the glucose transporter protein from cytosol to the cell surface. Glucose
transport protein carries out the facilitated transport of glucose.
3. Insulin stimulates phosphatase activity which removes phosphates from molecules activated by the kinase
cascade. Thus, insulin opposes the effects of glucagon and epinephrine.

Insulin also stimulates fatty acid biosynthesis as follows:

1. Insulin favors entry of glucose into cells, which, in turn, favors production of NADPH via entry of glucose-
6-phosphate into the pentose phosphate pathway.
2. Insulin activates the pyruvate dehydrogenase complex, which favors production of acetyl-CoA.
3. Insulin tends to reverse the effects of the kinase cascade, and stimulates dephosphorylation of acetyl-CoA
carboxylase, which favors polymerization of the
enzyme in an active form.

33
Figure 18.34: Regulation of fatty acid
synthesis in animal cells, such as liver cells.

Control of Fatty Acid Synthesis

Fatty acid biosynthesis is regulated largely by hormonal mechanisms. Acetyl-CoA

carboxylase, the first enzyme in the pathway, is an important regulatory enzyme for
the entire pathway. Fatty acid biosynthesis is inactivated in two ways through
control of acetyl-CoA carboxylase activity:
1. Phosphorylation of acetyl-CoA carboxylase by the cAMP-dependent protein
kinase tends to inactivate acetyl-CoA carboxylase by favoring depolymerization to
the monomeric form.
2. Long chain fatty acyl-CoAs inactivate acetyl-CoA carboxylase.

Conversely, fatty actid biosynthesis can be activated by insulin as follows:

1. Insulin promotes entry of glucose into cells which, in turn, favors production of
NADPH via entry of glucose-6-phosphate into the pentose phosphate pathway.
2. Insulin activates the pyruvate dehydrogenase complex, which promotes
production of acetyl-CoA.
3. Insulin reverses the effects of the kinase cascade, and stimulates
dephosphorylation of acetyl-CoA carboxylase. This, in turn, promotes the
conversion of the enzyme to its active, polymeric form.
In addition to hormonal/covalent regulation of acetyl-CoA carboxylase, allosteric
interactions of the enzyme with citrate or acyl-CoAs favor polymerization or
depolymerization, respectively, of the
enzyme.
All of the phosphorylation events are reversible
through the action of specific phosphatases.
 DM tipo I: insulino dipendente (10%)

DM tipo II: insulino-indipendente (90%)

insulino resistenza:

Anomalia del prodotto di secrezione

Antagonisti in circolo
Anomalie tessuti bersagllio (down-regulation)

D gestazionale (<15%)

aumento NEFA

[glu]s>160-180 mg/100ml
 emivita 5 min
fegato
adipociti

37
 glucagone

Ormone anoressizzante
(bassa affinità per
GlucagoneR) Tissue specific post-tranlational processing

GLP-1 (30 AA) rilascio dopo i pasti:

• Secrezione ridotta nel digiuno PC: proproteine convertasi
• Modula appetito Utilizzo per la cura del diabete? GRPP: glicentin related polypeptide
• Stimola la secrezione di INS e IP: interventing polypeptide-1
inibisce quella di glucagone GLP: glucagon like peptide
 emivita 5 min
fegato
adipociti

39
 Immagazzinato in granuli secretori che vengono rilasciati con meccanismo
inverso a quello dell’INS: elevate [ATP] inibiscono il rilascio, mentre un rapporto
ATP/ADP basso attiva ADC (cAMP) con conseguente attivazione di PKA e
apertura dei canali del Ca++

Target del glucagone:

• fegato (eff maggiore dell’adrenalina)

PEPCK
CAT-I
• adipociti (eff minore dell’adrenalina) G6PP

lipolisi

Secrezione amplificata da adrenalina che inibisce il rilascio di INS in quanto

a-cell ricche di recettori b-adrenergici, mentre b-cell di recettori a-adrenergici

Metabolizzato in fegato e rene

Responsabile dell’iperglicemia a digiuno nei casi di diabete grave

Muscolo
Glucagone fegato

FP-1 FP-2: fosfotreonine-fosfoserine

fosfatasi di tipo I e tipo II
 Somatostatina (cellule d isole pancreas)
ma anche ipotalamo e epitelio intestinale

Precursore 116 AA (preprosomatostatina)

Somatostatina 14

Somatostatina 28 (som 14 x 2)

Secrezione stimolata da glucosio, Arg e Leu

Riduce le secrezioni nel tratto gastrointestinale con rallentamento del

transito e riduzione dell’assorbimento dei nutrienti

Azione di inibizione sulla secrezione di INS e Glucagone

Nell’ipotalamo e nell’ipofisi ant. riduce la secrezione di GH eTSH

Recettor: GPCR con Gai

 adipochine
Leptina proteina (167 AA) secreta dal tessuto adiposo bianco (ma anche
stomaco e tessuti fetali)
Recettori ipotalamici (sazietà), muscolari e sistema immunitario
Concentrazione circolante correlata alla massa grassa (maggiore
nell’obesità)
Influisce sulla secrezione di INS e regola il metabolismo glucidico

Adiponectina proteina (224 AA, 30kDa) prodotta dai depositi viscerali

Molto abbondante nel torrente ematico
Recettori (adipoR1 adipoR2) presenti in muscolo, fegato, tessuto adiposo
Livelli più bassi nei soggetti obesi

Visfatina proteina (491 AA) prodotta dai depositi viscerali

Livello circolante proporzionale all’obesità addominale
Sembra legare recettori per INS con conseguente fosforilazione di IRS-1

Resistina, vaspina…
 Gastrin
Heptadecapeptide secreted by the antral mucosa of
stomach
• Gastrin stimulates acid secretion in stomach
• Product of preprogastrin - 101-104 residues
• Signal peptide cleavage leaves progastrin - 80-83
residues
• Cleavage at Lys and Arg (basic) residues and C-
terminal amidation leaves gastrin
• N-terminal residue of gastrin is pyroglutamate
• C-terminal amidation involves destruction of Gly
 Ormoni IPOTALAMICI

6 fattori di rilascio 3 fattori di inibizione:

CRF (corticotropina) SRIF (somatotropina)
TRF (tireotropina) PRIF (prolattina)
SRF (somatotropina) MRIF (melanotropina)
PRF (prolattina)
FSHRF (follicolo stimolante)
LHRF (luteinizzante)

Ipofisi anteriore
 Ormoni IPOFISARI

TSH (tireotropina): 2 subunità – a (la stessa di LH e FSH) attivante la adenilato

ciclasi e b per il riconoscimento dei recettori. Agisce sulla tiroide e sul tessuto
adiposo (lipolisi). Rilascio inibito per feedback da T3 e T4

GH (somatotropina): 191 AA stimola la sintesi proteica, è stimolata da SRF e

[Hys] e inibita da SRIF (somatomedine come IGF-I e IGF-II mediano alcune
sue funzioni) – recettori TyrK-JAK2-STAT

PRL (prolattina): 199 AA stimola sviluppo e secrezione della ghiandola

mammaria nella montata lattea, la sintesi di a-lattalbumina (lattosio sintasi),
G6PDH e 6-PGDH (via PP) – recettore come per GH

FSH (follicolo stimolante) maturazione dei follicoli ovarici, secrezione estrogeni

(f) e spermatogenesi (m)

LH (luteinizzante) stimola ovulazione e produzione di progesterone (f) e

testosterone (m)
 ATCH (corticotropina)

mediante cAMP
Sintesi del
pregnolone
Ormoni
corticosteroidi Rilascio di acidi
grassi dal TA
Recettori oppiodi
Ormone stimolante (analgesic effect)
i melanociti
 glucagone

Ormone anoressizzante
(bassa affinità per
GlucagoneR) Tissue specific post-tranlational processing

GLP-1 (30 AA) rilascio dopo i pasti:

• Secrezione ridotta nel digiuno PC: proproteine convertasi
• Modula appetito Utilizzo per la cura del diabete? GRPP: glicentin related polypeptide
• Stimola la secrezione di INS e IP: interventing polypeptide-1
inibisce quella di glucagone GLP: glucagon like peptide
 Immagazzinato in granuli secretori che vengono rilasciati con meccanismo
inverso a quello dell’INS: elevate [ATP] inibiscono il rilascio, mentre un rapporto
ATP/ADP basso attiva ADC (cAMP) con conseguente attivazione di PKA e
apertura dei canali del Ca++

Target del glucagone:

• fegato (eff maggiore dell’adrenalina)

PEPCK
CAT-I
• adipociti (eff minore dell’adrenalina) G6PP

lipolisi

Secrezione amplificata da adrenalina che inibisce il rilascio di INS in quanto

a-cell ricche di recettori b-adrenergici, mentre b-cell di recettori a-adrenergici

Metabolizzato in fegato e rene

Responsabile dell’iperglicemia a digiuno nei casi di diabete grave

While previous studies have investigated genetic variability in the
proglucagon gene across species (18), no studies have examined the
prevalence and spectrum of human genetic variation in the proglucagon
gene and its derived peptides within the human population…
 In the early 1980s proglucagon amino acid
sequences were first determined from anglerfish
isolates, followed by hamster and human cDNAs

Glucagon is thought to have originated first around 1

billion years ago with GLP-1 and GLP-2 created about
300 million years later by exon triplication of ancestral
glucagon

The glycogenolytic function of glucagon and its role

as a central hormone in blood glucose homeostasis
is preserved in all vertebrates - from jawless fish to
primates. This is reflected in its considerable
sequence conservation, with more than 72%
similarity to human for the most known divergent
sequence, the sea lamprey

jawless fish

Loss-of function mutations are often

deleterious and hence retained at very low
frequencies in the human population
 we analyzed the genetic
variation with respect to their
genetic location by mapping
all missense variants across
the 180 amino acid
preproglucagon sequence

the glucagon variant

R70HGlucagon,18 are
among the most
frequent, respectively
occurring 1 in 33,282
and 1 in 23,951
individuals.

Out of 29 positions, 23 amino acids in glucagon were found to display genetic

variants. Energy calculations revealed the variants T57IGlucagon,5, F58LGlucagon,6,
Y62CGlucagon,10, S63RGlucagon,11, Y65CGlucagon,13, D67AGlucagon,15 and
R70PGlucagon,18 to be highly destabilizing for the glucagon ligand-receptor
complex.
 Missense mutations found in the proglucagon-
derived peptides may not just impact receptor
affinity but may show altered expression and
secretion into the extracellular domain, altered
selectivity between receptors, changed kinetics
or internalization rates, switch modality or shift G
protein signaling selectivity.
Surprisingly, no disease-associated mutations, such as
from genome-wide association studies (GWAS), have
been identified within the coding region of GCG.
However, a mutation in the dipeptidyl-peptidase 4 (DPP-
4), which cleaves and inactivates GLP-1 and GLP-2,
has been shown to negatively affect glucosestimulated
GLP-1 levels, insulin secretion, and glucose tolerance
(84).
It also underlines that associations are difficult to identify
given confounding factors such as age, gender, life-style
factors, BMI, disease heterogeneity, and the impact of
environmental exposures. Moreover, disease associated
variants are less likely to occur in lowly and sparsely
expressed proteins such as GCG (87). In addition, most of
the GCG missense variants are very rare, not on commonly
used genotype arrays, and hence below GWAS detection-
threshold (88)
 ATCH (corticotropina) Ipofisi (a,i), cervello,
polmoni, linfociti,
placenta, gut

Produzione di ATCH è stimolata

da CRF ipotalamico e
angiotensina II

Ipofisi intermedia

mediante cAMP
Sintesi del
pregnolone
Ormoni
corticosteroidi Rilascio di acidi
grassi dal TA
Recettori oppiodi
Ormone stimolante i (analgesic effect)
melanociti (prodotto
dall’ipofisi intermedia)
 Ormoni che regolano il metabolismo del calcio:

Paratormone

Calcitonina omeostasi del Ca

Alterazioni
calcitriolo

ossa, denti
SN ipo: tetania iper: brachicardia
muscoli
coagulazione
trasduzione

Calcio 1 kg:
Calcemia 10mg/100ml • 1 g tessuti
Fosfatemia 5mg/100ml • 500 mg sangue
• >99% ossa
[Ca3(PO4)2]3 . Ca(OH)2 (idrossiapatite)
Perdite ca 2-400 mg/die
 Paratormone PTH (84 AA)
Pre-proparatormone

Azione ipercalcemizzante

Proteolisi selettiva (Golgi)

Bassa [Ca2+]
ematica
Secreto dalle paratiroidi

tubuli renali: prevenzione del

riassorbimento di Pi con conseguente
ipofosfatemia

richiamo di Pi dalle ossa

dove è legato al Ca2+ che
quindi viene liberato

ossa via cAMP rene via cAMP

mobilizzazione del Ca2+ stimola riassorbimento del Ca2+
stimola formazione calcitriolo - 25(OH)D3 idrossilasi

intestino Regolazione
maggior assorbimento del Ca2+ per feedback
 Calcitonina
Azione ipocalcemizzante
Secreto dalle cellule C parafollicolari della tiroide

calcemia

32 AA, come per PTH

proteolisi selettiva nel Golgi

Riduce il rilascio di Ca2+ e Pi dalle ossa

Aumenta l’escrezione di Ca2+ e Pi a livello renale

Non influenza assorbimento intestinale di Ca2+

 calcitriolo Azione ipercalcemizzante
dieta
Vit D2 (ergocalciferolo)
Trasportata nel sangue
da una globulina

microsomiale

calcidiolo
controllo da PTH

calcitriolo
Eliminazione
con la bile
vita media 24h

AZIONI:
Ossa: mobilizzazione dell’idrossiapatite

Intestino: aumenta assorbimento del Ca2+

(mediante aumentata espressione della
proteina legante il Ca2+)

Rene: riassorbimento Ca2+ e Pi

Pancreas: rilascio di INS

Deficienza: rachitismo (bambini) osteomalacia (adulti)

 Ormoni del tratto gastro-intestinale

nutrienti
peptidi secreti da cellule endocrine o neuroni del SN enterico condizioni (pH,
distensione…)
ormoni
struttura primaria spesso simile NT

regolano:
• secrezione acqua, enzimi, muco, ormoni
• motilità delle pareti
• crescita cellulare
• afflusso di sangue
• altri effetti su organi differenti (es. cervello
per sazietà e fame)

degradazione da peptidasi (fegato e rene)

 Ormoni del tratto gastro-intestinale
• gastrina (stimolata da AA, peptidi): secrezione di HCl e pepsina

• colecistochinina (stimolata da LCFA, MAG): contrazione della cistifellea e

secrezione succo pancreatico (induzione senso di sazietà)

• VIP (pep. vaso-attivo int.): rilascio muscolatura liscia, stimolazione

secrezione di acqua e elettroliti, stimolazione secrezione endocrina
pancreas, ipofisi, corteccia surrenalica; lipolisi e glicogenolisi nel fegato

• secretina (prodotta nell’intestino, stimolata da acidificazione): rallenta la

peristalsi e favorisce la secrezione pancreatica di acqua e HCO3-

• motilina: starter per le onde di contrazione muscolare

• neurotensina (stimolata da TG): inibisce secrezione gastrica e pancreatica

• sostanza P: contrazione muscolatura liscia, secrezione pancreatica,

vasodilatazione

• grelina: azione oressizzante (stimolo appetito) aumenta prima dei pasti e

decresce dopo di questi
cellule endocrine
neuroni
• peptide inibitore gastrico, enteroglucagone….
 Ormoni ipofisi posteriore

vasopressina (adiuretina, AVP) 9AA

ormone antidiuretico stimola il riassorbimento di

acqua nei tubuli contorti distali e connettori del
rene via GPCR (Gas, cAMP)
Azione vasopressoria mediante meccanismo
IP3 e DAG
Diabete Insipido (deficit di ormone), DI nefrogenico (neurone)
 Ormoni ipofisi posteriore
Ossitocina 9AA (7 uguali a AVP)

come per AVP

Sintetizzato come preproossitocina

(con neurofisina I)

Farmaco di
elezione per DI

Stimola la contrazione della muscolatura liscia: intestino crasso, vescica cistifellea, UTERO
(induzione parto)

Azione galattogoga (secrezione latte) per contrazione della muscolatura dei dotti

Azione mediata da IP3 e DAG

Ma anche…
 The renin-angiotensin system

regulation of blood pressure and

electrolyte metabolism (through
production of aldosterone)

Ansiotensinogeno: a2 globulina
prodotta dal fegato

Renina: prodotta dalle cellule

iuxtaglomerulari arteriola afferente
(barocettori e rec b-adr SNC)
k
- bac
f eed
r
e pe
iz ion
b
ini
angiotensin-converting enzyme (ACE)
Plasma, polmoni, cell. endoteliali

ACE inhibitors per ipertensione

stimola la secrezione di aldosterone (corticale)

 ormoni tiroidei

tetraiodotironina (T4)

triiodotironina (T3)

+I-
MIT
tireoglobulina (glicoproteina con 115 residui di Tyr)
DIT
 TSH

Model of iodide metabolism in the thyroid

follicle. A follicular cell is shown facing the
follicular lumen (top) and the extracellular
space (bottom). Iodide enters the thyroid
primarily through a transporter (bottom left).
Thyroid hormone synthesis occurs in the
follicular space through a series of reactions,
many of which are peroxidase-mediated.
Thyroid hormones, stored in the colloid in the
follicular space, are released from thyroglobulin
by hydrolysis inside the thyroid cell. (DIT,
diiodotyrosine; MIT, monoiodotyrosine; Tgb,
thyroglobulin; T3, triiodothyronine; T4,
tetraiodothyronine) Asterisks indicate steps or
processes where inherited enzyme deficiencies
cause congenital goiter and often result in
hypothyroidism

NIS: natrium/iodine symporter

trasporto nel sangue mediante:
TBG (thyroxine binding globulin) 70%
TBPA (thyroxine binding pre-albumin) 10%
albumina
 trasporto nel sangue mediante:
TBG (thyroxine binding globulin) 70%
in circolo T3 circa 1/5 di T4 TBPA (thyroxine binding pre-albumin) 10%
albumina

anabolico (stimolazione di sintesi proteica) GA3PDH

Na+/K+ ATPasi
catabolico (aumento consumo di O2 e UCP2-UCP3
produzione di calore)

metabolismo basale

T4 deiodinasi associata al RE T3
forma più attiva

rimozione ad opera del fegato mediante glucuronazione e solfatazione

eliminazione con la bile

distretti periferici tetraiodotireoacetico (TETRAC) e TRIAC

 Ormoni Steroidei

Surrenalici Sessuali
Corticosteroidi Maschili (testosterone..)
Mineralcorticoidi Femminili (estradiolo…)
Androgeni surrenalici

Derivano dal colesterolo

attivazione mediante fosforilazione (cAMP) di

STAR (STeroidogenic Acute Regulatory Protein)

migrazione CHO mitocondrio P450scc

IMM
 desmolasi e monossigenasi (NADPH e O2) sono indotte da:
ATCH (surrene)
LH (gonadi)

3bDH
via STAR D4,5isomerasi

trasportato nel sangue dalla

transcortina

metabolizzato dal fegato

(inattivazione)

utilizzo come contraccettivo

Pregnani C21

Androstani C19

Estrani C18
 St. Surrenalici
glucocorticoidi
mineralcorticoidi
androgeni surrenalici

Controllo ATCH (via cAMP)

altri animali Pathways involved in the
synthesis of the three major
classes of adrenal steroids
(mineralocorticoids,
glucocorticoids, and androgens).
glucocorticoidi

17α-hydroxylase and 17,20-lyase

uomo activities are both part of one
enzyme, designated P450c17.

Controllo renina-angiotensina by Ang II (Gq)

mineralcorticoidi
induzione di PC, PEPCK, FRU1,6DPP, GLU6PP
catabolismo proteico (muscolo scheletrico) e lipolisi

Trasportato da CBP
(transcortina) di
sintesi epatica, la
forma libera (8%) ha
attività biologica

induce trascrizione di
mRNA per
trasportatori del Na+
nei tubuli renali

cotrasporto Na+ e Cl-

carenza = eliminazione di Na+ e H2O con conseguente concentrazione

del sangue
tutti enzimi citoplasmatici
trasportato nel sangue da b-globulina (SHBP)

nel RE degli organi bersaglio (prostata, vescicole

seminali) si trasforma in DHT (diidrotestosterone)
per 5areduttasi NADPH dipendente

forma più attiva

esclusiva azione androgenica:

no estrogeni
no feedback su secrezione LH

Controllo da gonadotropine (LH, FSH)

metabolizzato nel fegato in androsterone e eliminato con bile e urine

RE
ormoni amminici
 catecolammine

tappa limitante
cAMP +

Secreti in granuli
Locus coeruleus (SN)
Substantia nigra (SN)
PLP
Midollare surrene

Cu - AscA O2

+ S-adenosilmetionina
glucocorticoidi

accumulate in granuli cromaffini (cromogranina A, ATP, ioni..) GPCR a (1-2) e b (1-2-3)

 azione Adr e NAdr b bloccanti

stimolazione glicogenolisi epatica

stimolazione glicogenolisi muscolare (differentemente da glucagone)

stimolazione lipolisi e aumento di NEFA plasmatici b adrenergici

aumento della gittata cardiaca cAMP

aumento della pressione arteriosa

+
vasocostrizione adenilato ciclasi
-
contrazione utero a2 adrenergici

diolatazione pupilla cAMP

a1 adrenergici: IP3 e Ca2+
disattivazione

Catechol-O-Methyl
Transferase (COMT)
HO HO
COMT
HO CHCH2 NHR' CH3 O CHCH2 NHR'
SAM S-Adenosyl-
Active R homocysteine R
catecholamine Inactive
metabolite
• COMT found in cytoplasm
• Terminates activity of catecholamines
• Catecholamine excretion products result from
combined actions of MAO and COMT
• Inhibitors of COMT (e.g., tolcapone) useful
in Parkinson’s disease 35
catecol-O-metiltransferasi (S-adenosilmetionina)

inattivazione deaminazione
acido 3-metossi-4idrossimandelico
MAO eliminato con le urine
 eicosanoidi (AA C20) e docosanoidi (DHA, C22)

Arachidonic acid is DHA (ω3, 22:6), is synthesized

present in membranes to a limited extent from α-
and accounts for 5% to linolenic acid or obtained
15% of the fatty acids in directly from fish oils, present
phospholipids in high concentrations in
retina, cerebral cortex, testis,
and sperm. DHA is particularly
needed for development of the
brain and retina and is
supplied via the placenta and
milk. Patients with retinitis
Prostaglandine pigmentosa are reported to
Prostacicline have low blood levels of DHA.

Trombossani
Leucotrieni
Lipossine
Resolvine Resolvine
Protettine
Neuroprotettine
LOX
leucotrieni:
infiammazione
acuta (edema)
e cronica

FANS

Steroidi PLA2

COX (en. Suicida)

Prostanoidi:
• PG
• prostacicline
• trombossani
Eicosanoidi:
Vita molto breve (inattivazione in situ per riduzione dei doppi legami, o rapido
catabolismo polmonare)

funzioni:

PG: mediatori dell’infiammazione, regolazione temperatura (PGF2),

Muscolatura liscia - vaso e branco dilatatione (PGE, PGA) o costrizione (PGF)
utero (stimolo-inibizione della contrazione uterina)
Modulatori dell’azione ormonale (mediante azione sull’ADC)
Nel SN modulazione dell’azione di NT (mediante azione sull’ADC)

PC e TX: azione antagonistica = TX (piastrine) az. aggregante, ipertensiva e

vasocostrittore, PC (endotelio) az. Antiaggregante, ipotensiva e vasodilatatrice

LT e LX: LT = chemotassi, reazioni allergiche, infiammazioni

LX = azione vasocostrittrice e ruolo immunosoppressivo

Resolvine: agiscono come risolutori del processo infiammatorio (riassorbimento

essudati, rimozione leucociti apoptotici) (azione mediata anche da protettine e
neuroprotettine)
BIOCHIMICA DEL SISTEMA NERVOSO
 Nervous
system

Central Peripheral
nervous system nervous system

Autonomic Somatic
Brain Spinal cord
nervous system nervous system

Parasympathetic Sympathetic
nervous system nervous system

craniosacrale toracolombare
Il SNC ha il compito di integrare
e coordinare le percezioni
sensoriali provenienti sia
dall’esterno che dall’interno del
corpo elaborando delle risposte
motorie che attivino o modulino
l’attività di specifici organi come
i muscoli o le ghiandole. Inoltre,
l’encefalo è la sede di funzioni
cognitive superiori

Il SNP ha il compito di veicolare le

percezioni sensoriali verso il SNC e
viceversa di portare dal SNC in
periferia comandi di tipo motorio.
The structure of the BRAIN
circa 1,4 Kg
volume circa 1200 cc
 Localisation of Function

is the idea that specific parts of the brain

have specific functions.

THE CEREBELLUM
! Balance
! Posture

! Cardiac, respiratory, and vasomotor centers

9
Hypothalamus
Moods and motivation
•Sexual maturation
•Temperature regulation
•Hormonal body processes

Pituitary Gland
•Hormonal body processes
•Physical maturation
•Growth (height and form)
•Sexual maturation
•Sexual functioning
Frontal Lobe

! Behaviour ! Inhibition
! Abstract thought ! Coordination of
processes movements
! Problem solving ! Generalized and mass
! Attention movements
! Creative thought ! Some eye movements
! Some emotion ! Sense of smell
! Intellect ! Muscle movements
! Reflection ! Skilled movements
! Judgment ! Some motor skills
! Initiative ! Physical reaction
! Libido (sexual urges)
11
Parietal Lobe

! Sense of touch ! Sensory

(tactile senstation) combination and
! Appreciation of comprehension
form through ! Some language
touch and reading
(stereognosis) functions
! Response to ! Some visual
internal stimuli functions
(proprioception)
 Temporal Lobe

•Auditory memories
•Some hearing
Occipital Lobe •Visual memories
•Some vision pathways
•Vision •Other memory
•Reading •Music
•Fear
•Some language
•Some speech
•Some behavior amd emotions
•Sense of identity
Attivazione del simpatico:
aumento di
• metabolismo basale
• stato di allarme e di
attenzione dell’individuo,
• ritmo del respiro,
• pressione
• ritmo cardiaco
dilatazione vie aeree
attivazione delle ghiandole
sudoripare
riduzione attività apparato
digerente e urinario.
Attivazione parasimpatico:
riduzione di
metabolismo basale
ritmo cardiaco
pressione sanguigna
aumentata secrezione delle
ghiandole salivari e di quelle
annesse all’apparato
digerente
aumentato afflusso di sangue
all’apparato digerente
stimola la defecazione e la
minzione.
Neurons, Glial Cells &
Neurochemistry
The Neuron – look at the diagram in
the handout

17

Direction of impulse
 The neuron - some facts

! Thereare billions of neurons in the

nervous system

! 80% of neurons are in the brain

!A nerve is a ‘bundle’ of neurons

18
! There are 3 main kinds of neurons
» Sensory (afferent) neurons (pseudopolari
o a T)
» Motor (efferent) neurons
» Inter (connector neurons)

! They vary in size from 4 microns (0.004 mm) to 100

microns (0.1 mm) in diameter. Their length varies
from a fraction of an inch to several feet.

19
! Unlike most other cells, neurons
cannot re-grow after damage
(except neurons from the
hippocampus). Fortunately, there
are billions of neurons in the brain.
! Information is passes from neuron
to neuron by electrochemical
transmission.
20
 Structure of neurons

1. Cell body - contains nucleus

2. Nucleus – contains DNA
3. Dendrites – cell body
(receives information)
4. Receptors – receive information using a
chemical signal.
5. Axons – sends information
6. Axon hillock – junction between cell
body and axon
**Lowest threshold for action potential**
 7. Terminal (buttons or boutons) –
swelling on the surface (see
slide)
- Inside buttons are synaptic vesicles,
packaging of neurotransmitter
8. Myelin sheath – insulation for axons
- comprised of glial cells (see slide)
A. In CNS it’s Oligodendrocytes
B. In PNS it’s Schwann cells
9. Nodes of Ranvier – spaces between
myelinating cells along the axon
23
CNS
It has been estimated that
oligodendrocytes in the optic
nerve produce between 30
and 50 internodes of myelin
slow mitotic rate and a poor regenerative capacity
oligodendrocytes are among the most
vulnerable elements and the first to
degenerate
PNS
CNS myelin contains some unique proteins:

•Myelin Basic Protein (MBP) – a 30 kDa protein with

a possible structural role
•Proteolipid protein (PLP) – it was the initial myelin
antigen, stabilize the dense line of myelin
•2’-3’cyclic nucleotide 3’ phosphodiesterase – to
date unknown function
•Myelin associated glycoprotein (MAG) - suggested
to have a role into signalling mechanisms between axon
and oligodentrocytes

MBP + PLP = 60-80% of total myelin protein

 astrocyte:
involvement in transport mechanisms, the
BBB system

Their major role is related to a connective

tissue or skeletal function since they
invest, possibly sustain and provide a
packing for other CNS components

a well-known function of the astrocyte is

concerned with repair. Subsequent to trauma,
astrocytes invariably proliferate, swell,
accumulate glycogen and undergo fibrosis by the
accumulation of filaments, expressed
neurochemically as an increase in glial fibrillary
acidic protein (GFAP).

the astrocyte is probably the most disease-

resistant component in the CNS because very
few diseases, other than alcoholism, cause
depletion of astrocytes
The microglial cell plays a role in phagocytosis
and inflammatory responses….

being able to become a very mobile, active

macrophage during disease and the major
effector cell in immune-mediated damage in the
CNS.
3. Synapse
- the junction between cells (neurons).
- synaptic cleft - space between cells
a. synapse is made of 3 parts:
1. Presynaptic cell– sending side of
synapse
2. Postsynaptic – receiving side of neuron
3. Synaptic Cleft
b. Purpose: promote chemical-electrical signal
c. Types of Synapses: axodendritic, axosomatic
axoaxonic, dendrodendritic
Neurons:Glia = 1:10
 Membrane Proteins
! Channels
! Pumps
! active transport

! Receptor protein sites

! bind messenger molecules

! Transducer proteins:
! 2d messenger systems

! Structural proteins
! form junctions with other neurons ~
Membrane Proteins: Ionophores
! Ions Channels
! Nongated
always open
! Gated
chemically-gated
electrically-gated
mechanically-gated ~
 Chemically-Gated Channels
! ligand-gated
! Ionotropic
receptor protein = channel
direct control ---> fast
! Metabotropic
second messenger system
indirect ---> slow ~
 Metabolic pumps
! Membrane proteins
! Pump ions
require energy
! Na+ - K+

! Ca++ (calcium)
! Also various molecules
! nutrients

! neurotransmitters ~
 Metabolic Pumps
! Active Transport mechanisms
Require energy
! Move materials against gradients
Na+ - K+
Calcium - Ca++
Nutrients, etc. ~
Two Types of Receptors (more again – soon)
1. Ionotropic Receptors
fast acting
ion channel/receptor complex same
only a few Neurotransmitter activate
them
2. Metabotropic Receptors
slower acting
ion channel and receptor are different
most Neurotransmitters act on them
12. Cytoskeletons (Neurofilaments) inside
cell
provide structural support
- Microfilaments
- Microtubules – Fairly large, play
important role in transport
a. Send vesicles to the buttons where
they are filled with NT. Acts like a
conveyor belt.
4. Chemical Milieu of Cellular Spaces when the
neuron is “at rest”
Intracellular space & extra cellular space
(inside of cell membrane & outside of cell
membrane)
a. Cl- = Chloride (more outside than inside)
b. Na+ = Sodium (more outside than inside)
c. A- = Anions (more inside than outside)
d. K+ = Potassium (more inside than outside)
Forces that maintain the chemical balance
i. Concentration gradient – lesser concentration
ii. Electrostatic pressure – attraction toward
opposite charges
iii. Na & K pumps – Throws out sodium and
takes in potassium to keep cell balanced
5. Four states of neuronal electrical charge
(potentials)
a. Resting Membrane Potential
-70 mV (transient state, constantly affected
by forces that increase or decrease
charge)
b. Excitatory Post-Synaptic Potential or EPSP–
Charge across the membrane becomes less
negative
- depolarization of the neuron (i.e. decrease
negative charge from –70mV to –65mV)
- Leads to an action potential
c. Inhibitory Postsynaptic Potential or IPSP
Charge across the membrane becomes more
negative
- hyperpolarization of neurons (i.e. increase in
negative charge from –70mV to –90 mV)
- Reduces the likelihood of an action potential
d. Action Potential or AP
Charge across the membrane becomes less
negative
- depolarization of neurons (i.e. decrease in
negative charge from –65mV to +55 mV)
- charge for the AP begins at Axon Hillock
- significant shift in ions
Cell membrane
structures and
functions
Membrane Transport
 Passive Diffusion
No special proteins needed
• Transported species simply moves down its
concentration gradient - from high [c] to
low [c]
• High permeability coefficients usually mean
that passive diffusion is not the whole story
 Facilitated Diffusion
DG negative, but proteins assist
• Solutes only move in the thermodynamically
favored direction
• But proteins may "facilitate" transport,
increasing the rates of transport
• Two important distinguising features:
– solute flows only in the favored direction
– transport displays saturation kinetics
 Active Transport Systems
Energy input drives transport
• Some transport must occur such that solutes
flow against thermodynamic potential
• Energy input drives transport
• Energy source and transport machinery are
"coupled"
• Energy source may be ATP, light or a
concentration gradient
 The Sodium Pump

aka Na,K-ATPase
• Large protein - 120 kD and 35 kD subunits
• Maintains intracellular Na low and K high
• Crucial for all organs, but especially for neural
tissue and the brain
• ATP hydrolysis drives Na out and K in
• Alpha subunit has ten transmembrane helices
with large cytoplasmic domain
 Na,K Transport
• ATP hydrolysis occurs via an E-P intermediate
• Mechanism involves two enzyme
conformations known as E1 and E2
• Cardiac glycosides inhibit by binding to
outside
 Calcium pumps

• Calcium levels in resting muscle cytoplasm are

maintained low by Ca-ATPase (PMCA) - a Ca pump
• Calcium is pumped into the sarcoplasmic reticulum
(SR) by a 110 kD protein that is very similar to the
alpha subunit of Na,K-ATPase (SERCA)
• Aspartyl phosphate E-P intermediate is at Asp-351
and Ca-pump also fits the E1-E2 model
VoV1-proton pumps

In neurons, they generate

the electrochemical
gradient that energizes the
H+-antiporters that load NT
into presynaptic vesicles
Biochemistry 2/e - Garrett & Grisham

Secondary Active Transport

Transport processes driven by ion
gradients
• Many amino acids and sugars are
accumulated by cells in transport
processes driven by ion gradients
• Na-dependent neurotrasmitters
recovery by glial cells and neurons

Copyright © 1999 by Harcourt Brace &

Biochemistry 2/e - Garrett & Grisham

Secondary Active Transport

• Symport - ion and the amino acid or
sugar are transported in the same
direction across the membrane
• Antiport - ion and transported species
move in opposite directions

Copyright © 1999 by Harcourt Brace &

 Resting Membrane Potential
• Membrane potential at which neuron
membrane is at rest, ie does not fire action
potential
• Written as Vr
Generation of Resting Membrane
Potential (-70mV)
• Plasma membrane
• Selective permeability, permeable to K, not
Na
• Unequal distribution of ions across
membrane
– Due to open potassium channels and closed
sodium and chloride channels
• Action of ion pumps 3Na/2K ATPase
 Definition
V=IR V=voltage, I=current, R=resistance

• g=1/R g=conductance

• Vm=membrane voltage

• Vr=voltage of membrane at rest

Ion Inside Outside Cross PM
K+ 125 5 yes
Na+ 12 120 no
Cl- 5 125 yes
H2O 55,000 55,000 yes
Anion- 108 0 no
 Ionic Equilibrium Potential

• The membrane potential that balances the

ions concentration gradient so that there is
no net current for that ion.

• No permeability factor.
 Equilibrium Potential of An Ion
• The membrane potential at which the net
driving force propelling the ion in = the net
driving force propelling the ion out.
• Written Eion; ENa, ECl, EK
 Nernst Equation
• Eion = 2.303 RT/zF log [ion]o/[ion]in

• Eion = ionic equilibrium potential

• Z= charge of ion
• F= Faraday’s constant
• T= absolute temperature (0Kelvin/-273°C)
• R= gas constant
 Permeability and Conductance
• gna is low at Vr because sodium
channels are closed
• gk is higher than gna at Vr because some
potassium channels are open.

• V=I/R Ohms Law

• G=conductance=1/R
 Definitions
• Current=net flow of • Resistance- frictional
ions per unit time forces that resists
• 1 ampere of current movement of ions or
represents movement charges
of 1 coulomb of • Measured in ohm
charge per second
• Current (A)= V/R
 Definitions
• Conductance is the • Capacitance refers to the
ability of plasma
reciprocal of membrane to store or
resistance and separate charges of
measures the ease with opposite signs.
which current flows in • Myelin has high
capacitance so stores
an object. charges and ions do not
• Measured in siemens move across the
(S) membrane
• Measured in Farads
 Ionic Equilibrium Potential

• Membrane Potential (potential difference

across the plasma membrane) at which the
net flow of an ion type = zero
• The number of ions moving into the cell =
the number of ions moving out of the cell
for a particular species of ion
 Nernst Equation Variables
• Assumes that membrane is permeable to
that ion
• As temperature increases the diffusion
increases
• As charge on the molecule increases, it
decreases the potential differences needed
to balance diffusion forces.
 Simplified Eion (at 37°C)
•Eion = 2.303 RT/zF log [ion]o/[ion]in

• ENa = 61.54mV log [Na]o/[Na]I = 62 mV

• EK = 61.54mV log [K]o/[K]I = -80 mV

• ECa = 30.77mV log [Ca]o/[Ca]I = 123 mV

• CCl = -61.54mV log [Cl]o/[Cl]I = - 65 mV

 Goldman Equation

• Vr= RT/F ln Pk[K]o+Pna[Na]o+PCl[Cl]i

Pk[K]I+Pna[Na]I+PCl[Cl]o

Also known as the constant field equation

because it assumes that electrical field of
the membrane potential is equal across the
span of the membrane
 Goldman Equation
•Vr= RT/F ln Pk[K]o+ Pna[Na]o+ PCl[Cl]i
Pk[K]I+ Pna[Na]I+ PCl[Cl]o

• Eion = 2.303 RT/zF log Pk[K]o+Pna[Na]o

Pk[K]I+Pna[Na]I

• Vr= 61.54 mV log 50[5]o +1[150]o

50[100]i+1[15]I

• = - 65mV
 Membrane Permeability
• Membrane is 50 more permeable to K than
to Na
• Pk/Pna = 50
• PCl/Pk = 0

• The membrane is so impermeable to

Chloride that you drop it from the equation
 Action Potential

Changes in Ion Permeability allows inward Na

flux and triggers an increased outward K flux
through voltage gated ion channels

Causes transient change in Membrane Potential

The change in ion permeability is triggered by

transient depolarization of the membrane
 Information Coding
• Is NOT in shape of action potential
• Is in the action potential frequency of firing
—how many are triggered
• In the action potentials pattern or timing of
propagation
 Conductance = g
• How many charges (ions) enters or
leaves cell (inverse of resistance)
• due to:
– number of channels/membrane area
• Highest density at axon hillock
– number of open channels
– ion concentration on either side of
membrane
– Measured in Siemens (S), in cells pS (pico; -12)
Action Potential: a transient and
rapid sequence of changes in the
membrane potential

Action Potentials
Can travel up to
100 meters/second

Usually 10-20 m/s

0.1sec delay between
muscle and sensory neuron
action potential
 Membrane Permeability
• Membrane is 50 more permeable to K than
to Na
• Pk/Pna = 50
• PCl/Pk = 0

• The membrane is so impermeable to

Chloride that you drop it from the equation
 Goldman Equation
•Vr= RT/F ln Pk[K]o+ Pna[Na]o+ PCl[Cl]i
Pk[K]I+ Pna[Na]I+ PCl[Cl]o

• Eion = 2.303 RT/zF log Pk[K]o+Pna[Na]o

Pk[K]I+Pna[Na]I

• Vr= 61.54 mV log 50[5]o +1[150]o

50[100]i+1[15]I

• = - 65mV
 Ion Permeability
• Changes during action potential

• The plasma membrane becomes permeable

to sodium ions
– Permeability increases from 0.02 to 20=1000
fold increase

• Causes Em aka Vr to approach Ena at positive

voltages = +20mV
 overshoot

Falling

rising

undershoot
 6 Characteristics of an Action
Potential
• #1 Triggered by depolarization

• a less negative membrane potential that

occurs transiently

• Understand depolarization, repolarization

and hyperpolarization
 #2 Threshold
• Threshold depolarization needed to trigger
the action potential

• 10-20 mV depolarization must occur to

trigger action potential
 #3 All or None
• Are all-or- none event
• Amplitude of AP is the same regardless of
whether the depolarizing event was weak
(+20mV) or strong (+40mV).
 #4 No Change in Size

• Propagates without The shape (amplitude &

decrement along time) of the action
axon potential does not change
as it travels along the axon
 #5 Reverses Polarity
• At peak of action potential the membrane
potential reverses polarity
• Becomes positive inside as predicted by the
Ena Called OVERSHOOT

• Return to membrane potential to a more

negative potential than at rest
• Called UNDERSHOOT
 #6 Refractory Period
• Absolute refractory period follows an action
potential. Lasts 1 msec

• During this time another action potential

CANNOT be fired even if there is a
transient depolarization.
• Limits firing rate to 1000AP/sec
 Stimulating electrode:
Introduces current that can
depolarize or
hyper-polarize

Recording electrode:
Records change in
Potential of the membrane
At a distance away
At Threshold Na influx equals K efflux

Voltage (mVolts) along Y axis

Time (msec)
 Voltage Sensitive Ion Channels
• Sodium
• Potassium
 Outline
! Why ion channels?
! Channel structure
! Ion channels have three basic functional properties
" Conduct
" Select
" Gate
! Evolutionary relationships between ion channels
! Various factors contribute to ion channel diversity
Channels are Made Up of Subunits
 Outline
! Why ion channels?
! Channel structure
! Ion channels have three basic functional properties
" Conduct
" Select
" Gate
! Evolutionary relationships between ion channels
! Various factors contribute to ion channel diversity
 Conduction
•Ion Channels Conduct Up to 108 Ions/sec

•Ion Channels Act As Catalysts

•Speed up fluxes

•Do not impart energy

•Driving force is provided by

electrochemical potential
 Outline
! Why ion channels?
! Channel structure
! Ion channels have three basic functional properties
! Conduct
! Select
! Gate
! Evolutionary relationships between ion channels
! Various factors contribute to ion channel diversity
Ion Channels are Selectively Permeable

Cation Permeable Anion Permeable

Na+ Cl -
K+
Ca++
Na+, Ca++, K+
 Outline
! Why ion channels?
! Channel structure
! Ion channels have three basic functional properties
! Conduct
! Select
! Gate
! Evolutionary relationships between ion channels
! Various factors contribute to ion channel diversity
Single Channel Openings are All-or-None in Amplitude,
With Stochastically Distributed Open and Closed Times

Closed

Open

2 pA
20 msec
There are Two Major Types of
Gating Actions
Gating Can Involve Conformational Changes
Along the Channel Walls
Gating Can Involve Plugging the Channel
 Gating Can Result from Plugging by
Cytoplasmic or Extracellular Gating Particles
There are Five Types of
Gating Controls
1) Ligand Binding

Extracellular

Cytoplasmic
2) Phosphorylation
 3) Voltage-gated

Change Membrane Potential

4) Mechanical Force-Gated
Stretch
 5) Temperature-gated

Current

Cold-Sensitive Heat-Sensitive

20 25 30 35 40 45 50

Temperature (º C.)
Modifiers of Channel Gating
Binding of Exogenous Ligands Can Block Gating
(Curare)

(BTx)

(ACh)
Ion Permeation Can be Prevented by
Pore Blockers

PCP

Glutamate-Activated Channel
Exogenous Modulators Can Modify the Action of
Endogenous Regulators
Current

Time

Open

Closed

Open

Closed
 Outline
! Why ion channels?
! Ion channels have three basic functional properties
! Conduct
! Select
! Gate
! Evolutionary relationships between ion channels
! Various factors contribute to ion channel diversity
 Ion Channel Gene Superfamilies

I) Channels Activated by Neurotransmitter-Binding

(pentameric channel structure):
•Acetylcholine
•GABA
•Glycine
•Serotonin

II) Channels Activated by ATP or Purine Nucleotide-

Binding (quatrameric or trimeric channel structure)
 Ion Channel Gene Superfamilies
III) Channels With Quatrameric Structure Related to
Voltage-Gated, Cation-Permeant Channels:
A) Voltage-gated:
•K+ permeant
•Na+ permeant
•Ca++ permeant
•Cation non-specific-permeant
B) Cyclic Nucleotide-Gated (Cation non-specific-
permeant)
C) TRP Family (Cation Non-specific); Gated by:
• osmolarity
• pH
• mechanical force (hearing, etc.)
• ligand binding
• temperature
D) Channels Activated by Glutamate-Binding
•quatrameric channel structure
•cation non-specific permeability
 Ion Channel Gene Superfamilies

IV) “CLC” Family of Cl--Permeant Channels (dimeric

structure):
Gated by:
•Voltage
•Cell Swelling
•pH

V) Gap Junction Channels (non-specific permeability;

hexameric structure)
 Outline
! Why ion channels?
! Channel structure
! Ion channels have three basic functional properties
! Conduct
! Select
! Gate
! Evolutionary relationships between ion channels
! Various factors contribute to ion channel diversity
Different Genes Encode Different
Pore-Forming Subunits
 Different Pore-Forming Subunits
Combine in Various Combinations
 The Same Pore-Forming Subunits Can
Combine with Different Accessory Subunits
Alternative Splicing of Pre-mRNA
 Ionic Equilibrium Potential

• Membrane Potential (potential difference

across the plasma membrane) at which the
net flow of an ion type = zero
• The number of ions moving into the cell =
the number of ions moving out of the cell
for a particular species of ion
Regenerative Process:

Once one Na channel

Opens, Na enters,
Depolarizes membrane,
More and more Na
Channels open leading to
More sodium influx &
causes upward &
depolarizing (more +)
phase of the AP
What does a sodium
Channel look like?

It is one large protein

With 4 domains that
Each loop through the
Plasma membrane 7
Times.
Na channel
 Property of Voltage Dependent
Sodium Channel
• Sodium channel opens for 1-2 millisecond
following threshold depolarization
• then inactivates and does not open even if
Vm is depolarized.
• This is called sodium channel inactivation
and contributes to the repolarization of Vm
Na Channel Gates
•M gate= activation gate
on Na channel; opens
quickly when membrane
is depolarized
•H gate- inactivation gate
on Na channel; Closes
slowly after membrane is
depolarized
•causes the absolute
refractory period for AP
propagation
 Potassium Channel Property
• K channels open with a delay and stay open
for length of depolarization
• Repolarize the Vm to Ek= -75mV which is
why you have hyperpolarization.

• Also called a delayed rectifier channel

Gate on the Delayed Rectifier
Potassium Channel
•K channels have a
single gate (n) that stays
open as long as Vm is
depolarized.
• n gate on K channels
opens very slowly this
allows the Vm to
depolarize due to Na
influx; Na and K
currents do not offset
each other right away
 Refractory Period
• Refractory period due to Na channel
inactivation and the high gK
• Subsequent Action potential cannot be
generated
 2 ways to increase AP
propagation speed
• Increase internal diameter of axon which
decreases the internal resistance to ion flow
• Increase the resistance of the plasma
membrane to charge flow by insulating it
with myelin.
 Channel Density
• Density is how many channels are in a unit
area of plasma membrane, ie how closely
they are packed together.
• Determines the length of the membrane that
will be depolarized at a given time
 Axonal transport
• Anterograde and retrograde
• It is the transport of substances within the
axon

• NOT to be confused with AP

 Anterograge axonal transport
• Substances are carried from the body of the
neuron toward the terminal vesicles in the
axon

• Substances: mitochondria, proteins,

neurotransmitters…
 Retrograde axonal transport
• Substances are transported from the
terminal vesicles of the axon toward the
body

• Examples: neurotransmitters, viruses

(rabies, herpes), toxin (tetanus)
 Synapses Between Two Neurons
• First neuron in path releases neurotransmitter onto
second neuron that responds to it
– 1st neuron is presynaptic neuron
– 2nd neuron is postsynaptic neuron
• Synapse may be axodendritic, axosomatic or
axoaxonic
• Number of synapses on postsynaptic cell variable
– 8000 on spinal motor neuron
– 100,000 on neuron in cerebellum
The Synapse
 Chemical Synapse Structure

• Presynaptic neurons have synaptic vesicles with

neurotransmitter and postsynaptic have receptors
 The Discovery of Neurotransmitters
• Histological observations revealed a 20 to 40 nm
gap between neurons (synaptic cleft)
• Otto Loewi (1873-1961) first to demonstrate
function of neurotransmitters at chemical synapse
– flooded exposed hearts of 2 frogs with saline
– stimulated vagus nerve of one frog --- heart slows
– removed saline from that frog & found it would slow
heart of 2nd frog --- “vagus substance” discovered
– later renamed acetylcholine
• Strictly electrical synapses do exist (gap junctions)
– cardiac & smooth muscle, some neurons & neuroglia
Otto Loewi experiment
 !"#$%&'()'!*)#+"(,'-.#)&/(&&(0)

!" #$%&'()*)+,%-+
)&./,0(
1" 2(3(,)(
4" 2(5(6&./+
,5&*7,&*.%
8" 9(:/.&/,%);*&&(/+
-(,5&*7,&*.%
sintesi e trasporto di NT classici e peptidici
3 diversi pool di vescicole sinaptiche nei neuroni:
• vescicole già ancorate alle zone attive (per rapido rilascio) 1-15%
• vescicole non a diretto contatto con PM (pool di riserva)
• pool di riposo (riserva funzionale)
 importanza del
meccanismo di
rilascio del NT

SNARE: SNAP REceptor

SNAP: Soluble NSF Attachment Protein

NSF: NEM sensitive factor

bersaglio di alcune tossine batteriche (cleavage di sinaptobrevina, SNAP25, sintaxina..)

tetaniche: paralisi spastica per inibizione
di rilascio di NT in interneuroni inibitori
botuliniche: paralisi flaccida per inibizione
di rilascio di ACh in motoneuroni
depolarizzazione determina
apertura dei VDC del Ca++
che si lega alle vescicole e
mediante legame con CaM
attiva la PKII che fosforila
la sinapsina I che
defosforilata manteneva
ancorate le vescicole
all’actina
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Ionotropic Receptor

Metabotropic Receptor
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 Types of Neurotransmitters
• 100 neurotransmitter types in 3
major categories
• Acetylcholine is formed from acetic
acid & choline
• Amino acid neurotransmitters
• Monoamines synthesized by
replacing -COOH in amino acids
with another functional group
– catecholamines (epinephrine,
norepinephrine & dopamine)
– indolamines (serotonin & histamine)
 Neuropeptide Classification
• Chains of 2 to 40 amino
acids that modify actions
of neurotransmitters
• Stored in axon terminal as
larger secretory granules
(called dense-core vesicles)
• May be released with
neurotransmitter or only
under stronger stimulation
• Some released from nonneural tissue
– gut-brain peptides cause food cravings
 Ionic Synaptic Transmission

• Cholinergic synapse produces ionotropic effect

– nerve signal opens voltage-
gated calcium channels
– triggers release of ACh which
crosses synapse
– ACh receptors trigger opening
of Na+ channels producing
local potential (postsynaptic
potential)
– when reaches -55mV, triggers action potential to begin
– synaptic delay (0.5 msec) is time from arrival of nerve signal at
synapse to start of AP in postsynaptic cell
 Metabotrophic Synapse Transmission

• Neurotransmitter uses 2nd messenger such as cyclic AMP

to alter metabolism of postsynaptic cell
Cessation & Modification of the Signal
• Mechanisms to turn off stimulation
– diffusion of neurotransmitter away from synapse into ECF where
astrocytes return it to the neurons
– synaptic knob reabsorbs amino acids and monoamines by
endocytosis & breaks them down with monoamine oxidase
– acetylcholinesterase degrades ACh in the synaptic cleft
• choline reabsorbed & recycled
• Neuromodulators modify synaptic transmission
– raise or lower number of receptors
– alter neurotransmitter release, synthesis or breakdown
• nitric oxide stimulates neurotransmitter release
 Neural Integration
• More synapses a neuron has the greater its information-
processing capability
– cells in cerebral cortex with 40,000 synapses
– cerebral cortex estimated to contain 100 trillion synapses
• Chemical synapses are decision-making components of the
nervous system
– ability to process, store & recall information is due to neural
integration
• Neural integration is based on types of postsynaptic
potentials produced by neurotransmitters
 Postsynaptic Potentials
• Excitatory postsynaptic potentials (EPSP)
– a positive voltage change causing postsynaptic cell to be more
likely to fire
• result from Na+ flowing into the cell
– glutamate & aspartate are excitatory neurotransmitters
• Inhibitory postsynaptic potentials (IPSP)
– a negative voltage change causing postsynaptic cell to be less
likely to fire (hyperpolarize)
• result of Cl- flowing into the cell or K+ leaving the cell
– glycine & GABA are inhibitory neurotransmitters
• ACh & norepinephrine vary depending on cell
Summation of Postsynaptic Potentials
• Net postsynaptic potentials in the trigger zone
– whether neuron fires depends on net input of other cells
• typical EPSP has a voltage of 0.5 mV & lasts 20 msec
• a typical neuron would need 30 EPSPs to reach threshold
– temporal summation occurs
when single synapse receives
many EPSPs in a short period
of time

– spatial summation occurs when

single synapse receives many
EPSPs from many presynaptic
cells
Summation of EPSP’s
 Presynaptic Inhibition

• One presynaptic neuron suppresses another one.

– Neuron I releases inhibitory neurotransmitter GABA
• prevents voltage-gated calcium channels from opening in neuron S so it
releases less or no neurotransmitter onto neuron R and fails to stimulate it
 Neural Coding
• Qualitative information (salty or sweet) depends upon
which neurons are fired

More rapid firing

frequency

• Qualitative information depend on:

– strong stimuli excite different neurons (recruitment)
– stronger stimuli causes a more rapid firing rate
• CNS judges stimulus strength from firing frequency of sensory neurons
– 600 action potentials/sec instead of 6 per second
 Neuronal Pools and Circuits
• Neuronal pool is 1000’s to millions of interneurons that
share a specific body function
– control rhythm of breathing

• Facilitated versus discharge zones

– in discharge zone, a single cell can produce firing
– in facilitated zone, single cell can only make it easier for the
postsynaptic cell to fire
Neural Transmission – the action potential

191
Action Potential: a transient
and rapid sequence of
changes in the membrane
potential

Action Potentials
Can travel up to
100 meters/second

Usually 10-20 m/s

0.1sec delay between
muscle and sensory neuron
action potential
All or None Law

Axons either "fire" or they don't "fire" to use the metaphor

of a gun. If they do "fire," then the size and speed
(amplitude and velocity) of the resulting action potential
along an axon is relatively invariant. Stronger stimuli do
not increase either the size or the speed. This is what the
"All or None Law" states.

How, then, does a neuron signal a stimulus of stronger

intensity? By firing more frequently. Frequency (i.e., the
number of action potentials per second) conveys the
strength of the original stimulus while each action
potential moves at the same speed and has the same
"size". 193
The Na+ and K+ channels
are also called voltage-
activated gates because
they act only when the
surrounding area reaches
a certain voltage.

The point at which the Na+

channel opens (1 on
diagram) is called the
threshold of excitation
(about -40 mV).

When the sodium ions

pour into the neuron (1 to 2
on diagram), only a very
small percentage of ions
actually do so. The
concentration of Na+ ions
inside increases by only
about 1%.
What are some real-world implications of this?

Some nerve poisons (e.g., scorpion venom) open Na+

channels and shut K+ channels & disrupts any action
potentials.

Local anesthetic drugs (Novocain, Xylocaine) block the

Na+ channels and prevent action potentials along
sensory neurons.

General anesthetics used in hospitals (ether,

chloroform) open some K+ channels in the brain a bit
wider than usual. This counter-acts the effects of Na+
channels being opened and prevents action potentials
from propagating, too. 195
So far, we've looked at an action potential as
it is generated at one place: the axon hillock.

How does it propagate or "move" down the

axon?

196
Look at the diagram to
the left. An action
potential propagates
along an axon by a
process in which,
once triggered, the
influx of positive ions
causes the adjacent
Na+ gate to open and,
in turn, this causes the
next Na+ gate to open,
and so on. Hence, an
action potential is
actually self-
propagating.
How fast does an action potential move along an
axon?

The thinnest axons propagate an action potential at

less than 1 meter per second (1 m/s).

Thick axons propagate action potentials at about 10

m/s.

However, myelin sheaths permit speeds up to 100

m/s.

HOW? 198
Saltatory conduction:
When the Na+ ions enter
the inside of the axon,
they quickly spread. The
insulation of the myelin
allows the ions to move
quickly to the next Na+
gate at a node of
Ranvier. And, so, the
action potential jumps
from one node to the
next.
How is a neuron triggered off and what
happens when the action potential reaches
the end of the axon?

! A typical neuron has about 1,000 to 10,000

synapses (that is, it communicates with 1,000-
10,000 other neurons, muscle cells, glands,
etc.).
! A neuron will ‘add up’ the input from these
synapses, some will be stimulating the neuron
and others will be inhibiting the neuron. Only
if the THRESHOLD of stimulation is reached, 200

will the neuron fire.

1. The transmitter substance is made and stored in
the vesicles
2. The Action Potential arrives at the synapse.
3. The Transmitter substance is released from the
vesicles.
4. The Transmitter substance flows across the
synaptic cleft.
5. The Transmitter substance attaches to the
appropriate receptor sites on the post synaptic
membrane (lock & key principle).
6. The post synaptic neuron summates the excitatory
and inhibitory input in order to decide whether or
not to fire.
7. The Transmitter substance is reabsorbed into the
vesicles of the synaptic button.
201
 Neurotrasmettitori

eccitatori inibitori
Glu GABA
Asp Gly
 Glutamate

• Most important for brain function

– Over half of the synapses are glutamatergic
– Glutamate released during injury is toxic in high amounts
• Despite it is the AA most present in diet, it does not cross BBB
– Synthesized in the brain from local precursors (Glucose)
• Packaged into vesicles by VGLUT
• Removed from the synaptic cleft by EAATs

• Con Asp rappresenta il 75% degli AA circolanti nel tessuto cerebrale

• coinvolto in tutte le aree encefaliche
• neuroni glutammatercici (movimento, apprendimento, memoria)
 Why Glutamate Receptors are
Important in Neurology:

Glutamate is present in millimolar quantities in most cells,

including neurons and glia
Glutamate is the main excitatory neurotransmitter in the
mammalian CNS
Glutamate is released in large quantities during
• Stroke
• Trauma
• Epilepsy
• Possibly in chronic neurological disorders
 Glutamate-glutamine Cycle
principale sistema di riciclo del Glu nel SNC

attenuazione/cessazione per ricaptazione del

Glu da parte di cellule pre- e perisinaptiche

non si conoscono altre vie per la demolizione

diretta per cui il ruolo della ricaptazione è
fondamentale per la modulazione dei segnali
eccitatori come anche per la eccitotossicità

trasportatori SLC6 family (Na/Cl dipendenti): dopamina (DAT), noadrenalina

(NET), serotonina (SERT), GABA (GAT1-2-3), Gly (Glyt1-2) (richiedono Cl- per
trasportare il NT)

trasportatori SLC1 family (Na/K) dipendenti: GlutT (EAAT)

 Gliotrasmissione…
gli astrociti non mostrano potenziale di azione
(potenziale di membrana stabile), ma possono:
• sintetizzare ed immagazzinare in vescicole
diversi NT (Glu, D-Ser, ATP, ANP, BDNF)
• rilasciare il contenuto a seguito di variazioni
di [Ca++] intracellulare
• sintetizzare il complesso proteico SNARE

rilascio del gliotrasmettitore ha azione paracrina (neurone pre e postsinaptico, astrociti).

Esempi:
• Secrezione del Glu ha azione sulla presinapsi (recettori metabotropici e ionotropici) con
stimolata liberazione del NT (e sincronizzazione dei neuroni)

• ATP rilasciato può agire su neuroni postsinaptici influenzando i livelli di espressione di

AMPAR
• adenosina agisce sui neuroni presinaptici iperpolarizzandoli (azione di inibizione)

possibile contributo dell’alterata gliotrasmissione nell’insorgenza di disturbi

neurologici (schizofrenia, epilessia…)
 Glutamate Receptors
• Ionotorpic receptors
– AMPA (a-amino-3-OH-5-metil-4-isossazolopropionico)
– Kainate—some are presynaptic and thought to regulate glutamate
release
– NMDA (N-metil-D-Asp) produces slower and long lasting currents
• Important in synaptic plasticity
- recettori d ???
azione inibitoria
solitamente
neurone
alta affinità presinaptico

passaggio non
selettivo di
Na+, K+, Ca++
azione
bassa affinità eccitatoria
neurone
postsinaptico
azione eccitatoria
 NMDA: localizzazione postsinaptica,
colocalizzano spesso con gli AMPAR,
elevata permeabilità al Ca++, in condizioni
di riposo sono bloccati da Mg++ la cui
inibizione è rimossa da depolarizzazione
(atività degli AMPAR)

AMPA: notevolmente espressi sui neuroni

postsinaptici, risposta eccitatoria rapida,
elavata permeabilità al Na+ e variabile al
Ca++

Kainato: localizzazione pre e

postsinaptica, attivazione rapida,
simili strutturalmente a AMPAR (possono
essere anche metabotropici – PLC)

RNA editing (mRNA): Arg invece di Gln nella subunità

GluR2 che conferisce impermeabilità al Ca++
 NMDA-R

D-Ser

alcol determina iperespressione di

questi recettori e la produzione
neuronale di Glu (dipendenza e
eccessivo tono glutammatergico)
 Recettori NMDA e metabotropici sono
implicati nel LTP (Long Term Potentioning)

apprendimento memoria

mediante PKC potenziano l’attività di NMDAR

 Learning
• Where does learning take place at the neuronal level? What do we mean
when we say neurons are plastic?

• Neurons are plastic at the synapses.

• Change in synaptic connectivity might constitute the mechanism of

learning

• "when an axon of cell A ... excite a cell B and repeatedly or persistently takes part in
firing it, some growth process or metabolic change takes place in one or both cells
such that A's efficiency, as one of the cells firing B, is increased" (Hebb, 1949)

• Cells that fire together, wire together

 Hebbian Learning
A B C
Pre-Synaptic
Neuron

Axon

Synaptic “Hebbian”
Cleft learning

Dendrite

Post-Synaptic
Neuron
 Mechanism for Hebbian Learning
• LTP is a candidate mechanism for Hebbian
learning (synaptic plasticity)

• LTP is a persistent increase in synaptic strength

(as measured by the amplitude of the EPSP)
that can be rapidly induced by brief neural
activity
 Where does LTP occur
• LTP has been found in mammalian neocortical regions, and in subcortical nuclei
• LTP has also been found in the peripheral nervous system of mammals
• LTP has mainly been studied in the hippocampus, a vital structure for memory

Properties of LTP
• Rapid
• Long Lasting effects
• Specificity
• Co-operative
• Associative
 How Does LTP Work?
• LTP requires some sort of additive effect
– High-frequency stimulation
• Activation of synapses and depolarization of
the postsynaptic neuron must occur at the
same time

• LTP additive effect takes place due to workings

of glutamate receptors
 Glutamate
• Glutamate is an excitatory neurotransmitter in CA1
 Vertebrate Models of Learning
• Synaptic Plasticity in the Hippocampus
Mechanisms of LTP in CA1
• Glutamate receptors mediate
excitatory synaptic transmission
– AMPARs
» Na+ ions enter to cause
EPSP
– NDMARs
» Ca++ entry only if
depolarized enough to
displace Mg++ ions that
clog channel
» Ca - PKC & CaMKII
» Inhibition of kinases
blocks LTP
– More AMPARs, more spines
 Accoppiamento Neurovascolare

• attività metaboliche molto variabili

• regolazione differenziale irrorazione per approvvigionamento di
nutrienti e carburante metabolico
• astrociti e sinapsi tripartita con azione sulle cellule endoteliali
• risposta emodinamica può essere misurata per scopi diagnostici
(fNMR)
accoppiamento neurovascolare
 GABA (acido g-aminobutirrico)
NT inibitorio

sinapsi tripartita: sintesi e regolazione

• principale NT inibitorio nel cervello dei mammiferi

• ruolo rilevante nel controllo di varie funzioni cerebrali e nella fisiopatologia di numerose
malattie mentali e neurologiche (discinesi del Parkinson e còrea di Hungtinton).

• non uniformemente distribuito a livello del SNC (maggiori concentrazioni sono state
riscontrate nella substantia nigra, nel globo pallido, nell’ipotalamo, nella corteccia, nel
cervelletto e nell’ippocampo);

• presente anche nelle cellule gliali.

 [GABA] dipende
dall’equilibrio tra rilascio
e ricaptazione ad opera di
GAT (SLC6 family)

GAD utilizza come cofattore il

PLP che deriva da vit B6
(piridossina), la cui carenza può
dare ipereccitabilità

nel SNP si può sintetizzare anche

attraverso una vie alternative a
partire dall’ornitina
• 35-40% of brain synapses use GABA
– Used in local circuit interneurons
– Used by Purkinje cells, a projection neuron
• Synthesis: glucose →glutamate →GABA
– GAD converts glutamate to GABA
• Transported into vesicles by VIAAT
• Inactivation by GAT in neurons and glia
• Degradation by mitochondrial enzymes solo una minima
parte, per il resto
viene riciclato tra
neuroni pre e
postsinaptici e
cellule gliali

acido valproico anticonvulsivante è inibitore della GABA-T

 recettori GABA

Ad oggi identificati 3 tipi di recettori per il GABA:

NOME EFFETTORE
• GABAA Canale per il Cl- target delle benzodiazepine e barbiturici (ansia, epilessia)

• GABAB Gi
(presinaptici)

• GABAC Canali per il Cl-

(neuroni della retina) potenziano il flusso di ioni
anche in assenza di GABA
sottoclasse di GABAA
favoriscono il legame del GABA
(modulazione allosterica positiva)
GABAA and GABAC are ionotropic
 GABAB
• presenti nel SN (neuroni e cellule gliali), nel sistema periferico sono presenti
nella muscolatura liscia e fegato

• metabotropico via Gai (inibizione lenta e prolungata della neurotrasmissione)

• GABAB1 subunità per i ligandi tramite dominio extracellulare e GABAB2

subunità accoppiata alla proteina Gi

• Gai inibisce la AC riducendo [cAMP], mentre la subunità Gbg attiva GIRK (G-
protein coupled rectified K-channel) e inibisce i VDCa-channel

• presenti nei neuroni presinaptici in neuroni GABAergici (autocettori) e non

GABAergici dove inibiscono (via riduzione della conduttanza del Ca++) il rilascio
di NT

• nei neuroni postsinaptici aumentano la conduttanza del K+ iperpolarizzando la

membrana e riduzione eccitabilità
 Glycine NT inibitorio
! 1965 discovery of its NT properties
! 50% of spinal cord synapses use glycine
! VIAAT transports glycine into vesicles
! Glycine transporters take up glycine (GlyT1 astrocyte,
GlyT2 neuron)
! Glycine receptors are ionotropic

! Mutations in the glycine transporter can result in

hyperglycinemia, a neonatal disease characterized
by lethargy and mental retardation.
Synthesis from serine
 canale del Cl-
Glycine receptor (ionotropico)

(pentamerico)

Dendrogram of Gly and

GABA iotropic receptors

consente l’ancoraggio ai
microfilamenti e una maggiore
concentrazione in siti specifici

Strychnine acts on glycine receptors — an antagonist

convulsioni e morte per blocco respiratorio
 Biogenic amines
tetrahydrobiopteran

• Regulate central and peripheral

homeostatic functions
• Affect a wide range of behavior
and are implicated in psychiatric
disorders
• Catecholamines
• Indoleamines (serotonin)
principali NT nelle terminazioni postgangliari SNP (ortosimpatico)
tappa limitante con TH attivata da (Ser) fosforilazione (PKA, ERK, CaMKII)
inibita dalle catecolammine
peculiarità (non ancora risolta)

associazione con proteine 14-3-3

 Dopamine (DA)
• Loaded into vesicles by VMAT
• Transported by DAT
• Inactivated in presynaptic cell
by MAO and COMT
• Metabotropic receptors

50-70 anni

NT più studiato (Parkinson, dipendenze)

Ruoli principali:
• controllo locomozione
• regolazione dinamiche rinforzo positivo (disfunzione = dipendenza patologica)

ruolo inibitorio del sistema motorio extrapiramidale

 DA Receptors (5 sottotipi)

D1-like D2-like
• D1 • D2
• D5 • D3
• D4
GPCR (Gas) GPCR (Gai)

[cAMP]

agonisti di DAR sono le anfetamine

ricaptazione circa 80%

Classification of Dopamine receptors
Catecholamines packed into
the synaptic vesicles

VMAT2:
Non-selective
and has high
affinity to
reserpine
target farmaci in patologie
neurodegenerative

analita valutato per il

metabolismo della
dopamina (Parkinson)
 Morbo di Parkinson e DA

• insorgenza 50-70 anni

• sintomatologia: tremore, lentezza dei movimenti, la cui comparsa
si ha con la perdita del 20-30% dei neuroni dopaminergici
(substantia nigra)
• comparsa di corpi di Lewi (accumuli di a-sinucleina)
• apparentemente una disfunzione mitocondriale potrebbe essere
correlata ad alcune forme di PD
• L-DOPA viene somministrato per il trattamento dei pazienti
(potenziamento dell’attività residua dei neuroni dopaminergici)
• DA non attraversa la barriera ematoencefalica
 Norepinephrine (NE)

principale NT del sistema ortosimpatico (organi

cuore, ghiandole, muscolatura vascolare e
alveolare) fight or flight response

nel SNC insieme ad altre monoammine regola le

funzioni superiori (umore, concentrazione,
motivazione)
 Norepinephrine (NE)
reserpina inibisce il
caricamento delle vescicole
! Loaded into vesicles by VMAT

! Transported by NET

! Inactivated in presynaptic cell by

MAO and COMT

! Metabotropic receptors

a1 (Gq), a2 (Gi) b1, b2, b3 (tutti Gs)

presenti solitamente nel neurone presinaptico (autocettori) per il controllo
della secrezione di NA

NE viene ricaptata ad opera di NET (Na-dipendente)

cocaina stimola il tono
noradrenergico
bloccando la
ricaptazione del NT

uso continuo determina

«affaticamento» dei
neuroni che riducono la
capacità di sintesi di
NE in assenza del
meccanismo di riciclo
catabolita escreto dai
reni e valutato nelle
urine per la diagnosi
di neoplasie endocrine
(feocromocitoma)
 Epinephrine (Epi)

• Loaded into vesicles by

VMAT
• No specific transporter
but NET is capable
• Metabotropic receptors
(gli stessi di NE)
 Serotonin (5-HT)

• uno dei NT più antichi

• regola diverse funzioni a livello centrale e periferico
• coinvolto nella regolazione di funzioni complesse quali
umore, funzioni cognitive, processi mnemonici

In the central nervous system, serotonin is believed to play an important role in

the regulation of body temperature, mood, sleep, vomiting, sexuality, and
appetite. Low levels of serotonin have been associated with several disorders,
namely clinical depression, obsessive-compulsive disorder (OCD), migraine,
irritable bowel syndrome, tinnitus, fibromyalgia, bipolar disorder, and anxiety
disorders. If neurons of the brainstem that make serotonin—serotonergic
neurons—are abnormal, there is a risk of sudden infant death syndrome (SIDS) in
an infant.
Serotonin (5-HT) indolamine
! Dietary tryptophan
! converted to 5–hydroxy– tryptophan by tryptophan hydroxylase
! then to 5-HT by a non–specific decarboxylase
! Specific transport system into cells
! Degradation
! mainly monoamine oxidase (MAO–A > MAO–B)
! 5–hydroxyindoleacetic acid (5-HIAA) in urine
tappa limitante con TrpH attivata da (Ser)
fosforilazione (PKA, ERK, CaMKII)
inibita dalle catecolammine, ma non da 5-HT

associazione con
proteine 14-3-3
 Serotonin (5-HT) indolamine

! Loaded into vesicles by

VMAT
! Transported by SERT
! Inactivated in
presynaptic cell by MAO
and COMT
! One ionotropic receptor
(5-HT3), the rest are
metabotropic
 Serotonin roles
! Central
! Peripheral
" control of appetite
" peristalsis
" sleep
" vomiting
" mood
" platelet aggregation and haemostasis
" hallucinations
" inflammatory mediator
" stereotyped behaviour
" sensitisation of nociceptors
" pain perception
" microvascular control
" vomiting
 Serotonin Receptors
! At least 15 types and subtypes
! Multiple transduction mechanisms
! 5HT-1A: role in anxiety/depression
! 5HT-1D: role in migraine
! 5HT-2: role in CNS various
behaviors, and in cardiovascular
system
! 5-HT3: role in nausea and vomiting
esp. due to Chemotherapy.
 Histamine
• Neurons that release histamine as their
neurotransmitter are located in the hypothalamus
and projects to the cerebral cortex, the limbic system
and thalamus.
• There are 3 types of histamine receptors, H1,H2, H3 .
• Anti allergic drugs act by blocking H1 receptors and
causes sedation.
• H3 receptors involved in vascular tone control.

• pazienti con Alzheimer presentano livelli ridotti di Ist

Istamina
viene ricaptata dagli astrociti

tessuti periferici

cervello
Main signaling pathways for
histamine receptors.
Histamine can couple to a
variety of G-protein-linked
signal transduction
pathways
via its four different
receptors. The H1 receptor
activates the
phosphatidylinositol
turnover via Gq/11
proteins. The other
receptors either
positively (H2 receptor) or
negatively (H3 and H4
receptor) regulate adenylyl
cyclase activity via Gs and
Gi/o protein activation
respectively.
Several additional signaling
pathways have been
described, which are not
shown. Abbreviations:
PIP2, phosphatidylinositol
4,5-bisphosphate;
PLC, phospholipase C; AC,
adenylyl cyclase; ATP,
adenosine triphosphate;
cAMP, cyclic AMP; PKC,
protein kinase C; PKA,
protein kinase A.
 Histamine receptor

implicati meccanismi temperatura corporea implicati meccanismi più abbondanti

sonno/veglia rilascio di prolattina sonno/veglia nei distretti
periferici
acetilcolina (ACh)
ChAT solubile più abbondante
ChAT associata alla membrana vescicola sinaptica
ACh è immagazzinata nelle vescicole attraverso VAChT

rimossa dall’attività della AChE presente sulla faccia esterna

della membrana postsinaptica

principale NT della placca neuromuscolare dove sono

presenti i recettori ionotropici (nicotinici, N1)

funzioni complesse nel SNC…nella malattia di Alzheimer

c’è una severa perdita di neuroni colinergici corticali
 Acetylcholine As a
Neurotransmitter

! Both the central and peripheral

nervous systems.
! Binds two broad classes of
receptors:
! Nicotinic receptors

! Muscarinic receptors.
 Nicotinic Receptor Features
! Composed of 5 subunits:
! 2 a, b, g and d.
! Subunits are arranged to form a
central cavity that extends across the
membrane.
! Nicotinic receptors are also channels
! ACh-binding opens gates and allows
ion fluxes across the channel
Figure 6: Nicotinic Receptor
Channel
Agonist
Binding
Site

Gate
 Subclasses of Nicotinic
Receptors
! Skeletal muscle (N1 or Nm)
! Unique a and b subunits

! Autonomic ganglia (N2 or Ng).

! Both N1 and N2 are gene-product
families not single receptor types.
Other Ligand-Gated Channels
! Structural and sequence similarity to
nicotinic receptors.
! Example agonists for these channels
include:
! Serotonin (5-HT)

! Glutamate

! GABA

! Glycine
 Muscarinic receptors
! Muscarinic receptors are not
channels.
! Operate through G-proteins to alter
second messenger systems.
! 5 muscarinic subtypes have been
cloned and sequenced (M1, M2, M3,
M4, M5).
 Grouping Muscarinic
Receptors
! M1, M3, and M5 receptors: Activate
Phospholipase C through Gq.
! PLC activation ---> increased IP3 --

> increased intracellular Ca++

! Increased intracellular Ca++ --->

Activation of Ca++-sensitive K+ &

Cl- channels.
 Grouping Muscarinic
Receptors

! M2 and M4 receptors
! Gi -coupled inhibition of adenylate

cyclase
! Go or Gi -coupled regulation of

certain Ca++ & K+ channels.

NEUROTRASMISSIONE
PURINERGICA
 Teoria purinergica

Inizialmente fu scarsamente considerata, per via della

enorme diffusione delle purine (ubiquitarie) che mal si
conciliava con eventi così specifici come i processi di
neurotrasmissione.
Successivamente, sono stati dimostrati numerosi ruoli
dell’adenosina e dell’ATP nel SNC e SNP, nel sistema
cardiovascolare, renale, respiratorio ed immunitario.
Inoltre, le purine sembrano svolgere un ruolo
neuroprotettivo.
Possono sia essere co-modulatori di altre
neurotrasmissioni (ad es. catecolamine e ATP sono
concentrati nelle vescicole in rapporto 4:1), che agire su
recettori propri.
Sintesi e metabolismo delle purine
Sistema Purinergico

The major pathways of

intracellular adenosine
metabolism
 Recettori Purinergici

A1 e A3: Gi
A2: Gs
A1 adenosine receptors are inhibitory in the central
nervous system.
Antagonisti di P1A1 sono caffeina, teofillina, teobromina
(aumento dell’attenzione e della concentrazione)

Risposte mediate da P2X sono di tipo rapido transiente ed

eccitatorio
Recettori dell’Adenosina
NEUROTRASMISSIONE
DA NEUROPETIDI
The number of
known
neuropeptides far
exceeds the number
of fast-acting
conventional
neurotransmitters
 Trasmissione da neuropeptidi oppiodi
Sintetizzati nel corpo cellulare, trasportati in periferia (trasporto
anterogrado) ai terminali dove sono rilasciati (accumulati in
concentrazione minore rispetto ai classici NT); non sono ricaptati,
ma le vescicole vengono recuperate (trasporto retrogrado):
sintesi e trasporto di NT classici e peptidici
 Neuropeptides are packaged into large, dense-core vesicles.

Diversity is generated by families of propeptides, alternative

splicing, proteolytic processing and posttranslational
modifications.
I neuropeptidi oppioidi sono sintetizzati a partire da precursori:

POMC (prepropiomelanocortina)
Proencefalina
Prodinorfina
Neuropeptidi
Neuropeptides may act directly on a postsynaptic target at the
synapse; presynaptically on the terminal that released the peptide
(autocrine effects); on an immediately adjacent cell (juxtacrine
effects); or on a cell a few cell diameters away from the site of
release (paracrine effects). In addition, peptides can exert their
actions after traveling through the circulation to reach their target
(endocrine effects), as in the case of hypothalamic releasing
factors and neurohypophyseal hormones.

Neuropeptides make a unique contribution to signaling:

the release of neuropeptides generally requires a more intense
stimulus than that required for release of fastacting classical
neurotransmitters.
Neuropeptides play key roles in appetite
regulation and obesity.

(Enkephalin knockout mice reach

adulthood and are healthy).

Neuropeptides are crucial to pain perception.

 Neuropeptidi oppiodi
I neuropeptidi oppioidi rilasciati attivano specifici recettori posti su
cellule bersaglio

Esistono diversi recettori per gli oppioidi distribuiti in varie e

specifiche parti del SNC e in numerosi organi periferici (intestino,
ghiandole endocrine, adipociti, ecc)

Tipi di recettori (tutti accoppiati a proteine Gi)

µ: morfina,
d: enchefaline,
k: dinorfina,
s: dinorfina

I più abbondanti sono i recettori di tipo µ

 OPPIO
L’oppio è conosciuto dall’epoca faraonica.
Sostanza medicamentosa occidentale portata in Oriente all’inizio del Medioevo; è ottenuto
incidendo le capsule immature del Papaver somniferum e raccogliendone la linfa che trasuda,
che poi viene lasciata rapprendere all'aria in una resina scura che viene impastata in pani di
colore bruno, che emanano un odore dolciastro e hanno un sapore amaro.

L’oppio contiene almeno 20 principi attivi, alcuni privi di attività narcotica

(papaverina)
Prima purificazione dei derivati fenantrenici nel 1806: morfina, codeina e tebaina
In seguito derivati semisintetici
 Neuropeptidi oppiodi

I farmaci oppiacei attivano (agonisti) o inibiscono (antagonisti)

i recettori per gli oppioidi

Gli effetti dei farmaci oppiacei riflettono le funzioni del

sistema oppioide nel nostro corpo
Effetti farmacologici degli oppiacei
Il dolore
Gli oppioidi hanno azione analgesica:

- riducendo il rilascio di trasmettitori

eccitatori da neuroni afferenti primari,
- inibiscono i neuroni che trasmettono il
dolore localizzati nel midollo spinale,
- attivano i neuroni inibitori del dolore
discendenti, mediante inibizione di
interneuroni GABAergici inibitori.
Effetti farmacologici degli oppiacei

Effetto analgesico

b-endorfina e dinorfina hanno capacità analgesica centinaia di volte

superiore alla morfina
Senza perdita di coscienza
Aumenta la soglia al dolore
Cambia la sensazione del dolore
Efficacia massima per dolori profondi, continui
 Effetti farmacologici degli oppiacei

Depressione respiratoria:
Diminuisce la frequenza del respiro e il volume respiratorio
Azione sui centri pontini e midollari
Riduzione risposte a CO2

Tosse:
Effetti inibitori a dosi più basse di quelle che causano inibizione
respiratoria
 Effetti farmacologici degli oppiacei
Tratto gastrointestinale:
- Antidiarroico
- Aumenta il tono e causa spasmo (non usare per il controllo di coliche
biliari)

Muscolature lisce
Pochi effetti in generale a dosi terapeutiche, ma possono:
- precipitare attacco asmatico
- prolungare travaglio
- causare anuresi

Sistema Cardiovascolare
- Calore e prurito facciale (liberazione di istamina)
- Ipotensione da depressione respiratoria a cui segue aumento della
produzione di liquor (non usare per traumi cranici)
 Effetti farmacologici degli oppiacei

Adattamenti alla somministrazione ripetuta dei farmaci

(oppiacei ma non solo)

La stimolazione prolungata di un recettore (farmaci agonisti) spesso

provoca riduzione della capacità di rispondere al farmaco
(tolleranza). Spesso è possibile superare la tolleranza aumentando la
dose somministrata

L’inibizione prolungata di un recettore (farmaci antagonisti) spesso

provoca un innalzamento della capacità di rispondere (ipersensibilità)
 Effetti farmacologici degli oppiacei

“TOLLERANZA” e “DIPENDENZA”

La TOLLERANZA è un evento biologico che avviene a

livello di singola cellula (anche se può poi avere
manifestazioni generali)

La DIPENDENZA è un evento biologico che coinvolge una

parte delle funzioni psichiche del SNC
Cannabinoidi
 Cannabinoids as Modulators of
Neuronal Function
OH

Exogenous Plant-derived Δ9-Tetrahydro-

cannabinoids (phytocannabinoids) cannabinol O

OH
Synthetic cannabinoids Nabilone

O
Endogenous Endocannabinoids Anandamide OH
cannabinoids NH

2-Arachidonoyl O
OH
glycerol O
OH

Pacher P et al. Pharmacol Rev. 2006;58:389-462.

Endogenous Cannabinoid Life
Cycle
! Biosynthesis-from membrane
phospholipids
! “On-demand” synthesis-not
stored like classical transmitters
 Cannabinoid Receptors
! Originally demonstrated with specific binding of
CP-55940; a synthetic cannabinoid agonist
! CB1 cloned in 1990; G-protein coupled; various
effects including inhibition of adenylate cyclase,
modulation of voltage-dependent calcium,
potassium channels
! CB1 mostly presynaptic (NT release inhibition?)
! CB2 40% sequence similarity; found in many
immune system cells
! CB1 knockouts eliminate most (but not all) of the
CNS effects of THC
NEUROTRASMISSIONE
NITRERGICA
 La scoperta dell’NO (EDRF)

Nel 1980 Furchgott e Zawadzki documentarono come la rimozione

dell’endotelio da preparati vascolari in vitro fosse in grado di prevenire
la vasodilatazione indotta da acetilcolina: fattore EDRF
Moncada nel 1987 scoprì che l’EDRF corrispondeva all’NO (fino ad
allora considerato solo un inquinante ambientale)
 La sintesi dell’NO
La sintesi di NO avviene per trasformazione enzimatica dell’arginina
operata dalla NO sintasi con produzione finale di citrullina:
NO synthesis and reactions
NO, a simple gas, is able to
diffuse across the membrane,
and alters the activity of
intracellular target enzymes.

NO reversibly binds to ferrous iron within

haemproteins, such as guanylate
cyclase, haemoglobin and cytochrome
oxidase. NO reacts at the diffusion
limited rate with superoxide to produce
peroxynitrite. NO or peroxynitrite, can
react with thiols to produce nitrosothiols.
 Nitric Oxide & free radical
Production

H 2O
SIDE
OU
T
Ca2+

e
E

as
I D
INS

tal
.
ca
. . SOD Fe 2+
O2- O2- H 2O 2 OH
Mitochondria

NO OONO -

NOS
Ca 2+-CaM ROS
[Ca2+]
PLA 2
Arachidonic acid COX Ca2+
Nitric Oxide Synthase Isoforms

NOS-1 (155 kDa): neuronal, brain, type I-NOS;

central and peripheral neurons, islet, endometrium,
skeletaal muscle

NOS-2 (125 kDa): inducible, type II-NOS;

macrophage, liver, smooth muscle endothelium, heart

NOS-3 (135 kDa): endothelial, type III-NOS;

endothelium, brain, heart
 Effetti dell’NO
L’NO prodotto dalle NOS I e III agisce essenzialmente su due
bersagli intracellulari:

1. attiva la guanilato ciclasi citosolica (inibizione della fosfolipasi

C con conseguente riduzione della concentrazione
intracellulare di calcio)
2. inibisce la citocromo c ossidasi (in caso di carenza di ossigeno,
impedisce il consumo del gas lungo la catena respiratoria)

L’NO prodotto dalla NOS II induce vasodilatazione (può portare a

shock endotossico non responsivo a sostanze vasocostrittrici),
facilita la proliferazione dei linfociti ed ha un’azione battericida
Rapporti tra NO e glutammato (azione transinaptica)
In seguito a depolarizzazione del neurone
presinaptico, il glutammato viene
rilasciato dalla terminazione nervosa ed
attiva i recettori non-NMDA presenti sulla
membrana del neurone postsinaptico e che
inducono depolarizzazione. La
depolarizzazione consente l’ingresso di
Ca2+ nel neurone attraverso i recettori di
tipo NMDA attivati dal glutammato.
L’aumento della [Ca2+] attiva la NO
sintasi neuronale che è Ca2+-calmodulina
dipendente. L’NO formato può sia agire a
livello della sede di formazione che
diffondere nello spazio extracellulare e
stimolare la guanilato ciclasi a livello
delle terminazioni nervose e della glia.
L’NO a livello presinaptico stimolerebbe
il rilascio del glutammato dalle vescicole.
 IV. Guanylate Cyclase:
cGMP and Nitric Oxide As
Second Messengers
 Nitric Oxide and cGMP

Intracellular
Ca++ Stores Ca++ C.M. Membrane Bound
Guanylate Cyclase
Ca++
NO GTP
Ca++ Soluble
NO
Synthetase
NO Guanylate Cyclase
+
Citrulline GTP PDE
cGMP GMP
Arginine
Ion Channels
cGMP-Dependent PK
PDEase Activity
guanine

cGMP
 Attivazione fosfodiesterasi

Attivazione cGMP dipendente di proteine kinasi

Inibizione fosfolipasi c (minor produzione IP3 e DAG)

Inibizioni recettori IP3

Modula negativamente l’effetto di aumento di
[Ca++] intracellulare in una sorta di feedback
desensitization of the β-adrenergic receptor
mediated by receptor phosphorylation.
 BRAIN METABOLISM

1. Energy metabolism of the brain

2. Ammonia handling
 Energy metabolism of the
brain
! 2% of body weight, 20% of energy
expenditure

! GLUCOSE is the main fuel (25% of

the whole body)
! daily consumption 120g

! adopted starvation (3 weeks):

oxidation of ketones in the brain

covers up to 50% of energy
accoppiamento neurometabolico
Corpi chetonici?
BLOOD BRAIN BARRIER
 Blood brain barrier
! History:
! 19th century, Ehrlich: aniline dye

i.v. stains all organs except brain

! 1960: morphology by electron

microscope
! Function:
! BBB selectivity protects the brain
 Blood brain barrier
! Morphology: endothelium, BM,
astrocyte
 Blood brain barrier selectivity
! Free permeability (passive diffusion):
! small molecules: H2O, O2, CO2, NH3,

ethanol
! lipid soluble molecules: steroid

hormones
! Carrier mediated transport:
! glucose: GLUT-1 (insulin independent)

! amino acids

! Pinocytosis
 Neuroni cerebrali: GluT3 (affinità maggiore per glucosio) rispetto a
GluT1

Manca nel cervello la fruttosio1,6DiPi fosfatasi …… non fa

gluconeogenesi!

Ipoglicemia = stato confusionale, successivamente coma

Glicogeno presente in quantità limitate (sufficienti solo per pochi minuti)

soprattutto negli astrociti

HK presente a livelli 20 volte maggiori rispetto agli altri tessuti

2 isoforme: una solubile nel citosol ed un’altra legata al mitocondrio (più
attiva)

Il legame HK-mitocondrio è inversamente proporzionale al rapporto

ATP/ADP
 What´s the first thing that
happens when you think?
! Excitatory firing ® Glu uptake by glia ® Na+
influx ® ATP consumption by Na-K-ATPase ®
activation of glycolysis ® lactate transported to
neurons

! Local increase in lactate increases blood flow

! Excitotoxity = excesive Glu release

! epilepsy, traumatic brain injury
+
! Na and Ca
2+ IC accumulation ® swelling
 Functional imaging of the
brain

! PET = positron emission tomography

!
18F-2-deoxy-2-fluoroglucose

! taken up by glia, phosphorylated

but not further metabolized

! active areas of the brain

accumulate tracer
 8-10% del glucosio prende la via dei
pentoso fosfati...

Tale shunt è minimo in condizioni di

maggiore richiesta di energia e massimo in
condizioni in cui c’è attiva sintesi lipidica
(sintesi della mielina)
Functional imaging: PET
 Oxygen uptake
! Brain: 20% of whole-body O2 consumption
! The most vulnerable to hypoxia
" 5 min of VF/arrest may lead to irreversible brain

damage
" temperature dependent
Ammonia handling in the brain
! NH3 is a waste product of deamination
reactions (Gln®Glu, Glu®2-OG etc.)
! Metabolism:
! Glutamin synthetase: NH3 + Glu ® Gln

! Gln is metabolized in the liver/kidneys

! Ammonia toxicity:
NH3 + 2OG + NADH ¨ Glu + NAD+
! Krebs cycle impairment: 2-OG depletion

! Glu excess, excitotoxicity

mitocondri neuronali:
varie…
Evidence has accumulated over the last two decades indicating that L-LAC is an important cerebral
oxidative-energy substrate (for refs. see [4,48,49]): brain can take up L-LAC from blood, particularly
during intense exercise, as well as in the initial minutes of recovery [50]. Moreover, an ‘‘astrocyte–
neuron L-LAC shuttle’’ has been proposed, in which astrocytes take up glucose from blood,
convert it into L-LAC via glycolysis and then export L-LAC into the extracellular phase via the
isoform 1 of monocarboxylate transporter (MCT1). In turn neurons take up extracellular L-LAC
via the isoform 2 of monocarboxylate transporter (MCT2) and use it as a fuel for mitochondrial
respiration [51].
Recently it was hypothesized that, in the brain, L-LAC is the principal product of glycolysis,
whether or not oxygen is present [34]. These observations again raise the question of mitochondrial
involvement in oxidation of L-LAC by brain and evidence presented in 1959 by Sacktor et al. [6]
suggests that this does indeed occur. After nearly 50 years, we have confirmed that neurons (in
particular cerebellar granule cells) can take up and metabolize externally added L-LAC. L-LAC
metabolism can occur also in mitochondria from these cells: the existence of an mL-LDH located in the
inner mitochondrial compartment was shown by both immunological and enzymatic analysis.
Consistently, externally added L-LAC enters mitochondria, perhaps via an L-LAC/H+ symporter,
and is oxidized in a manner stimulated by ADP [52]. As in [7,11,16], an L-LAC/PYR shuttle
operates in neurons (Scheme 2).
Whether these translocators are those found in mitochondrial reticulum by Hashimoto et al., who
showed the presence of mL-LDH and MCT1 and MCT2 in rat brain and primary neuron cultures [53],
remains to be investigated. Very recently in an astrocyte cancer cell line Lemire et al. showed good
evidence of the presence of mL-LDH [54]. This further confirms that intracellular L-LAC shuttles
could occur in both astrocytes and neurons.
L-Lactate metabolism in brain: astrocyte–
neuron shuttle, intracellular L-lactate
shuttle and the L-lactate/pyruvate shuttle.

The picture of L-LAC metabolism emerging from

some papers quoted in this minireview is as
follows. Astrocytes can produce L-LAC from
taken up glucose. L-LAC can be oxidized inside
mitochondria via the mL-LDH (Intracellular L-
lactate shuttle) and/or released in the extracellular
fluid from which it can be taken up by neurons
(astrocyte/neuron shuttle). Inside neuron, L-LAC
enters mitochondria via the L-LAC/H+ symporter
as well as in exchange with endogenous PYR
which originates in the matrix via the mL-LDH.
The newly synthesised PYR can in turn move in a
carrier-mediated manner to the extramitochondrial
phase where is converted to L-LAC (intracellular
L-lactate/pyruvate shuttle).
The release of cytochrome c from either
GEN- or DZN- S-K5 cell mitochondria.
(A) Cyt c release from S-K25 or S-K5
cells, in the absence or presence of GEN
and DZN (20 mM each), at 3 h after
apoptosis induction, was detected by
means of oxygen consumption caused by
externally added ASC (5 mM).
(B) Cyt c release from S-K25 or S-K5
cells, in the absence or presence of GEN
and DZN at the indicated concentrations,
at 3 h after apoptosis induction, was
detected as in (A) Results, expressed as
natoms O/min mg cell protein, are the
means S.D. of triplicate measurements
and are representative of at least six
different experiments carried out with
different cell preparations obtained from
different groups of animals.