Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
MOLECULAR CELL BIOLOGY SIXTH EDITION CHAPTER 11 Transmembrane Transport of Ions and Small Molecules
2008 W. H.Freeman and Company Copyright 2008 W. H. Freeman and Company
A study of mutant zebrafish with pale stripes led to the identification of a sodium/calcium transporter that regulates the darkness of human skin
The phospholipid bilayer is a barrier that controls the transport of molecules in and out of the cell.
Gases diffuse freely, no proteins required. Water diffuses fast enough that proteins arent required for transport
The bilayer is permeable to: Small hydrophobic molecules Small uncharged polar molecules The bilayer is impermeable to: Ions Large polar molecules THEREFORE, need membrane proteins to transport most molecules and all ions across biomembranes
KEY CONCEPTS Selective transport across the lipid membrane requires transport proteins Transport proteins are integral membrane proteins that move molecules and ions There are two classes of transport proteins: transporters (pumps) and channels
Also called
Na+/K+ ATPase
Three main class of membrane protein 1.ATP- power pump( carrier, permease) couple with energy source for active transport binding of specific solute to transporter which undergo conformation change 2. Channel protein (ion channel) formation of hydrophilic pore allow passive movement of small inorganic molecule 3. Transporters uniport symport antiport
Partition Coefficient
Permeability coefficients (in cm/sec) through synthetic lipid bilayers Product of the concentration difference (in mol/cm3) and permeability coefficient (in cm/sec) gives the flow of solute in moles per second per square centimeter of membrane
Cell membrane Barrier to the passage of most polar molecule Maintain concentration of solute Diffusion rate depends on : 1. Concentration gradient or electrochemical gradient 2. Hydrophobicity i.e. higher partition coefficient 3. Particle size
The rate at which a molecule diffuses across a synthetic lipid bilayer depend on its size and solubility The smaller the molecule and the less polar it is, the more rapidly it diffuses across the bilayer
Membrane proteins mediated transport of most molecules and all ions across biomembrane
Overview of membrane transport proteins 1. All transmembrane proteins 2. Some transport has ATP binding sites 3. Move molecules uphill () against its gradient
Differences 1. Transporters= -uniporters transport a single molecule down its gradient (passive) -co-transporters couple movement of a molecule down its gradient with moving a molecule up its gradient (active) 2. Pumps = hydrolyze ATP to move small molecules/ions up a concentration gradient or electric potential (active) 3. Channels = transport water/ions/small molecules down their concentration gradients or electric potentials (passive)
The four mechanisms of small molecules and ions are transported cross cellular membranes
Ion force
If transport substance carries a net charge, its movement is influenced by both its concentration gradient and the membrane potential, the electric potential (voltage) across the membrane. Substance concentration + electric potential = electrochemical gradient. Determines the energetically favorable direction of transport a charged molecule across a membrane.
+
Electrical gradient
(affects only charged solutes)
Electrochemical gradient
Copyright 2009, Dr. Salme Taagepera PhD. All rights reserved.
Two types of transport are defined by whether metabolic energy is expended to move a solute across the membrane.
Passive transport: no metabolic energy is needed because the solute is moving down its concentration gradient. In the case of an uncharged solute, the concentration of the solute on each side of the membrane dictates the direction of passive transport. Active transport: metabolic energy is used to transport a solute from the side of low concentration to the side of high concentration.
Facilitated Diffusion
Free Diffusion
A. Non-channel mediated lipids, gasses (O2, CO2), water B. Channel mediated ions, charged molecules
Facilitated diffusion
Carrier mediated glucose, amino acids
Facilitated Diffusion Rate of diffusion is determined by: 1. concentration gradient 2. amount of carrier protein 3. rate of association/dissociation
Glucose: utilize glucose as a source for ATP production Water: utilize aquaporins to increase the rate of water movement. H2O: hybrophilic, did across membrane
Families of GLUT proteins(1-12) Highly homologous in sequence, and contain 12 membrane-spanning helices. Different isoforms different cell type expression, and different function GLUT2: express in liver cell ( glucose storage) and cell( glucose uptake) pancreas GLUT4: found in intracellular membrane, increase expression by insulin for remove the glucose from blood to cell GLUT5: tansport fructose. Other isoforms: ???
intestine, lesser amounts few others fructose transport gluconeogenic tissues preimplantation blastocyst mediates flux across endoplasmic reticulum embryonic insulindependent transport
Oded Meyuhas
Each GLUT protein contains 12 membrane-spanning alpha helices Differentially expressed EXAMPLE: GLUT4 is only expressed in fat and muscle cells Fat and muscle cells respond to insulin by increasing their uptake of glucose, thereby removing glucose from the blood In absence of insulin = GLUT4 on intracellular membranes In presence of insulin = GLUT4 found on cell surface QUESTION: Defects in directing GLUT4 to the cell surface can cause what common disease? Type II diabetes, high blood glucose
Glucose
GLUT1 is responsible for transporting glucose across the bloodbrain barrier GLUT1 provides glucose for the brain GLUT1 deficiency syndrome: Brain does not obtain enough glucose from the blood Symptoms: seizures, developmental delay, motor disorders Treatment: ketogenic diet (high fat/low carb diet)
Copyright 2009, Dr. Salme Taagepera PhD. All rights reserved.
Liposome containing a single type of transport protein are very useful in studying functional properties of transport proteins
It is a major experimental tool to study the biochemistry of transport protein function in vitro Widely used as a drug delivery system and for gene transfection
Movement of water Osmosis: movement of water across semipermeable membrane Osmotic pressure: hydrostatic pressure uses to stop the net flow of water
Hypertonic solution: the concentration is higher than cytosol Isotonic solution: equal to cytosol Hypotonic solution: lower; and water move to cytosol Animal cell ace a problem in maintaining their cell volume within a limited range, thereby avoiding lysis Plant cell: has cell wall prevent cell shape Turgor pressure (): osmotic pressure, plasma membrane against water into the cytosol and then into the vacuole turgor pressure supplies rigidity The large forces of turgor pressure are resisted by the strength of cellulose microfibrils in the cell wall
Hypertonic external solution: concentration of water is low relative to its concentration inside the cell Water moves out down its concentration gradient and the cell shrinks Hypotonic external solution: concentration of water is high relative to its concentration inside the cell Water moves in down its concentration gradient and the cell swells
Water draw is equal inside and outside When the Na+ K+ pump stops, Na+ goes into the cell along its concentration gradient This adds to the solute concentration in the cytosol Water moves into the cell along its concentration gradient and the cell bursts
Aquaporins increase the water permeability of cell membrane Water cross membrane is very lower Water cross membrane via specific channel- aquaporins
Expression of aquaporin by frog oocytes increases their permeability Egg move to hypotonic environment
Injection aquaporin mRNA
control
kidney cells resorb water from urine; mutation diabetes insipidus large volume urine
2-nm-long water selective gate 0.28nm gate width Highly conserved arginine and histidine in the gate H2O for HO bonding with cystein
Aquaporins are membrane water channels that play critical roles in controlling the water contents of cells. Water crosses the hydrophobic membrane either by simple diffusion or through a facilitative transport mechanism mediated by these specialized proteins. These protein channels are widely distributed in all kingdoms of life, including bacteria, plants, and mammals. Important in osmotic regulation, acting to prevent bursting of the cells whenever there are changes of the exterior salt concentration.
Must phosphorylation
Transport process requires ATP hydrolysis in which the free energy is liberated by breakdown of ATP into ADP and phosphate.
inside
V-class H+ ATP ase pump protons across lysosomal and vacuolar membrane
inside
ATP powered pump 1. P- class 2, 2 subunit i.e. Na+-K+ ATP ase, Ca+ATP ase, H+pump 2. F-class locate on bacterial membrane , chloroplast and mitochondria pump proton from exoplasmic space to cytosolic for ATP synthesis 3. V-class maintain low pH in plant vacuole
ATP-powered ion pumps generate and maintain ionic gradients across cellular membranes
Extracellular intracellular
Extracellular intracellular
Neel large energy: RBC need 50% ATP for Na/K pump; nerve and kidney need 25% for ion transport
Muscle relaxation depends on Ca2+ APTase that pump Ca2+ from the cytosol into the sacroplasmic reticulum (SR)
In muscle cell, cytosol Ca2+ 10-7 M (resting state) to 10-6M (contraction) Most intracellular Ca2+ storage in SR(10-2M). Ca2+ is released from the sarcoplasmic reticulum through Ca2+ release channels when the muscle contracts Most cytosol Ca2+ transport into SR via Ca2+ ATPase pump. Ca2+ pump comprises 90% of the sarcoplasmic reticulum membrane protein Responsible for restoring the Ca2+ gradient (pumps it back into the sarcoplasmic reticulum
10-2
Higher Ca+2
10-6
Low affinity for calcium
Lower Ca+2
-helix
Conformational change
Calmodulin regulates the plasma membrane Ca2+ pump that control cytosolic Ca2+ concentration
Calmodulin-mediated activation of plasma membrane Ca2+ ATPase leads to rapid Ca2+ export keep cytosolic Ca2+ very low
Ca++ signal Ca
calmodulin
Ca++-release channel
++
endoplasmic reticulum
ATP
Ca ATP
Allosteric activation
signalactivated channel Ca++
Ca++-ATPase
Na+/K+ ATPase maintain the intracellular Na+ and K+ concentration in animal cell ATP-powered ion pumps generate and maintain ionic gradients across cellular membranes
Extracellular
intracellular
High Low
low high
Membrane potential
Na+/K+ ATPase Four major domains: M - Membrane-bound domain, which is composed of 10 transmembrane segments N- Nucleotide-binding domain, where adenine moiety of ATP and ADP binds P Phosphatase domain, which contains invariant Asp residue, which became phosphorylated during the ATP hydrolysis A domain essential for conformational transitions between E1 and E2 states
Na+- K+ Pump on the Plasma Membrane K+ is 10 to 20 X higher inside animal cells than outside Na+ is 10 to 20 X higher outside animal cells than inside These concentration gradients are maintained by the Na+ - K+ pump on the plasma membrane Pump operates as an antiporter, pumping K+ in and Na+ out Transport cycle depends on autophosphorylation of the protein Terminal phosphate of ATP is transferred to an aspartic acid of the pump Ion pumps that autophosphorylate are called P-type transport ATPases
The Na+- K+ pump is electrogenic + + It generates an electrical potential (known as membrane potential) across the membrane Reason: Pumps 3 Na+ ions out for every 2 K+ ions it pumps in Thus the inside of the cell is negative relative to the outside
Electrogenic effect of the pump contributes only ~10% of the membrane potential remaining 90% is only indirectly attributable to the Na+- K+ pump (discussed later)
ABC Transporters
Largest family of membrane transport proteins 78 genes (5% of genome) encode ABC transporters in E coli Many more in animal cells Known as the ABC transporter superfamily They use the energy derived from ATP hydrolysis to transport a variety of small molecules including: Amino acids, sugars, inorganic ions, peptides. ABC transporters also catalyze the flipping of lipids between monolayers in membranes All ABC transporters each contain 2 highly conserved ATPbinding domains
Bacterial permease are ABC proteins that import a variety of nutrients from the environment ATP-binding cassette
Structure of the E coil BtuCD protein, an ABC transporter mediating vitamin B12 uptake
Bacterial permeases are ABC proteins that import a variety of nutrients from the enviornment About 50 ABC small-molecule pumps are known in mammals ABC transporter 2 T ( transmembrane ) domain, each has 6 - helix form pathways for transported substance 2A ( ATP- binding domain) 30-40% homology for membranes
i.e. bacterial permease use ATP hydrolysis transport a.a ,sugars, vitamines, or peptides inducible, depend on the environmental condition i.e. mammalian ABC transporter ( Multi Drug Resistant) export drug from cytosol to extracellular medium mdr gene amplified by drugs stimulation mostly hydrophobic for MDR proteins cancer cell resistant to drug mechanisms
Auxiliary transport system associated with transport ATPases in bacteria with double membranes The transport ATPases belong to the ABC transporter supefamily
The approximately 50 mammalian ABC transporters play diverse and important roles in cell and organ physiology
A typical ABC transporter consists of four domains two highly hydrophobic domains and two ATP-binding catalytic domains
ATP binding leads to dimerization of the two ATP-binding domains and ATP hydrolysis leads to their dissociation.
ABC protein that transport lipid-soluble substrates may operated by a flippase mechanism
Structural model of E. coli lipid flippase, and ABC protein homologous to mammalian MDR1
Certain ABC proteins filp phospholipids and other lipid-soluble substrates from one membrane leaflet to opposite leaflet
Flippases
Lipids can be moved from one monolayer to the other by flippase proteins Some flippases operate passively and do not require an energy source Other flippases appear to operate actively and require the energy of hydrolysis of ATP Active flippases can generate membrane asymmetries
2.
3.
4.
5.
defect in ABC transport protein( ABCD1) located on peroxisome, used for transport for very long fatty acid; absence ABCD1 fatty acid accumulate cytosol cell damage 2. Tangiers disease
Dificiency in plasma ABCA1 proteins, which is used for transport of phospholipis and cholesterol 3. Cystic fibrosis
mutation of CTFR( cystic fibrosis transmenbrane regulator; a Cltransporter in the apical membrane of lung, sweat gland and pancrease)
licked it did not resorption of Cl taste saltyThis leads to abnormalities in the pancreas, skin, intestine, sweat glands and lungs
Flippases move phospholipids from one membrane leaflet to the opposite leaflet
asymmetric distribution of phospholipids senescence or apoptosis disturb the asymmetric distribution Phosphatidylserine (PS) and phosphatidylethanolamine: cytosolic leaflet exposure of these anionic phospholipids on the exoplasmic face signal for scavenger cells to remove and destroy Annexin V a protein that specifically binds to PS phospholipids fluorescently labeled annexin V to detect apoptotic cells flippase: ABC superfamily of small molecule pumps
Yeast sec mutant at nonpermissive temp: secretory vesicle cannot fuse with plasma membrane purify the secretory vesicles
(dithionite)
In vitro fluorescence quenching assay can detect phospholipid flippase activity of ABCB4
,! ------ , ,
59mv
Na ,
-59mv -59mv
K, ,
Negative charge on intracellular organic anions balanced by K+ High intracellular [K+] generated by Na+-K+ ATPase Large K+ concentration gradient ([K+]i:[K+]o 30) Plasma membrane contains spontaneously active K+ channels K+ move freely out of cell As K+ moves out of cell, leaves negative charge build up opposes further K+ exit At equilibrium, electrical force balances concentration gradient and electrochemical gradient for K+ is zero (even though there is still a very substantial K+ concentration gradient) Resting membrane potential = flow of positive/negative ions across plasma membrane precisely balanced Membrane potential measured as voltage difference across membrane For animal cells, resting membrane potential varies between -20 and -200 mV Negative value due to negativity of intracellular compartment compared to extracellular fluid Because K+ channels predominate in resting plasma membrane, resting membrane potential mainly due to K+ concentration gradient Nernst equation permits calculation of membrane potential (V):
Potential difference exists across every cells plasma membrane. cytoplasm side is negative pole, and extracellular fluid side is positive pole Inside of cell negatively charged because: large, negatively charged molecules are more abundant inside the cell sodium potassium ATPase pump resting K+ ion channels (from in to out flow)
Ion channels contain a selectivity filter formed from conserved transmembrane helices and P segment
Ion channels contain a selectivity filter formed from conserved transmembrane a helices and p segments Ion-selectivity filter
Transmembrane domain
Voltage-gated K+ channels have four subunits each containing six transmembrane helices
Ion channels have ion selectivity - they only allow passage of specific molecules Ion channels are not open continuously, conformational changes open and close
EACH OF THE binding sites closely mimics potassium ions' octahedral hydration shell, thereby minimizing the energy required to strip off their water coats. Because of their smaller size, sodium ions don't fit in these binding sites as snugly and thus find the energetic cost of trading their water coat for a spot in the selectivity filter too high.
In the vestibule, the ions are hydrated. In the selectivity filter, the carbonyl oxygens are placed precisely to accommodate a dehydrated K+ ion. The dehydration of the K+ ion requires energy, which is precisely balanced by the energy regained by the interaction of the ion with the carbonyl oxygens that serve as surrogate water molecules
effect
effect
V=I x R
Novel ion channels can be characterized by a combination of oocyte expression and path clamping
Na+ entry into mammalian cells has a negative change in free energy
Cotransport:
Use the energy stored in Na+ or H+ electrochemical gradient to power the transport of another substance Symport: the transportd molecules and cotransported ion move in the same direction Antiport: the transported molecules move in opposited direction Depending on how many solute molecules are transported and in what direction, carrier proteins are dubbed uniporters, symporters, or antiporters.
The differences in ion Electrochemical gradient: concentrations across the membrane establishes a membrane electrochemical gradient Membrane potential Ion concentration gradients across the membranes establishes the membrane electric potential
Concentration gradient
An electrochemical gradient combines the membrane potential and the concentration gradient
Na+ linked symporters import amino acids and glucose into animal cells against high concentration gradients Operation Model for the two-Na+/one glucose symport
Carrier oscillates between state A and state B Binding of Na+ and glucose is cooperative (binding of either ligand induces a conformational change that enhances binding of the 2nd ligand) Since Na+ higher in the extracellular space (& very low inside), glucose more likely to bind in A state Accordingly, Na+ and glucose enter the cell (by an A to B transition) more often than they leave the cell Result is net transport of Na+ and glucose into the cell
Three carrier proteins, appropriately positioned in the plasma membrane, function to transport glucose across the intestinal epithelium.
Increases PM area
More contraction
Carrier proteins in the plasma membrane regulate cytosolic pH (pHi) at about 7.2
Cotransporters that regulate cytosolic pH H2CO3 H+ H+ + HCO-
3. Na+/H+ antiport There are two mechanisms by which this pH is regulated - H+ is transported out of the cell Na+-H+ exchanger, an antiporter, couples the influx of Na+ to an efflux of H+ - HCO3- is brought into the cell to neutralize H+ in the cytosol Na+ -driven Cl- - HCO3- exchanger uses a combination of the two mechanisms by coupling an influx of Na+ and HCO3 to an efflux of Cl- and H+
The activity of membrane transport proteins that regulated the cytosolic pH of memmalian cells changes with pH
A putative cation exchange protein plays a key role in evolution of human skin pigmentation
pH 36
Low pH, maintained by V-class ATP-powered pump
Numerous transport proteins enable plant vacuoles to accumulate metabolites and ions
pyrophosphate-hydrolyzing proton pump (PPi -powered pump) The H+ pump inside inside positive powers mover negative ion move inside; High positive inside antiport many ion and sucrose inward
More positive
Trans-epithelial transport Import of molecules on the lumen side of intestinal epithelial cells and their export on the blood facing sides
Transcellular transport of glucose from the intestinal lumen into the blood
3
[high]
Basolateral Na+/ K+ ATPase generates Na+ gradient that drives the Symporter
microvilli
Parietal cells acidify the stomach contents while maintaining a neutral cytolic pH
Acidification of the stomach lumen by parietal cells in the gastric lining
3 1 P-class 2
END
END