NRSC500 Problem set September 2017

PROBLEM SET

OHM’S LAW: V = I x R or I = g x V

NERNST EQUATION: EK = (RT/zF) ln {[K +]OUT / [K +]IN}

where at T=200C, 2.3(RT/F) ~ 58 mV {z=1, ln = 2.3 x log10}

GOLDMAN-HODGKIN-KATZ EQUATION:
Vrev = (RT/F) ln ( PK[K+]o + PNa[Na+]o + PCl[Cl-]i) where z=1
( PK[K+]i + PNa[Na+]i + PCl[Cl-]o)

1) a. Which two factors determine the resting membrane potential in neurons?

b. Complete the following table by listing the typical millimolar concentrations for Na+, K+, Ca2+, and
Cl- outside and inside the neuron. What are the typical concentration gradients for these ions
(outside/inside)? What is the equilibrium potential for each ion, based on these concentrations?

ION [inside](mM) [outside](mM) Gradient Equilibrium potential (Ex )

2) Fill in the blank or circle the correct answer: [Note that the “resting state” refers to a stable membrane
potential in the absence of voltage clamp]

a. In the resting state, the neuronal membrane potential is in the range of ________ mV.

b. In the resting state, the most permeable ion across the neuronal membrane is _____.

c. In the resting state, there is no ion flow across the neuronal membrane – True or False? (circle
one)

d. In the resting state, the net ion flow across the neuronal membrane is inward / outward / zero
(circle one).

e. The Nernst equation describes equilibrium when the ____________ driving force is balanced by
the _____________ driving force.
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NRSC500 Problem set September 2017

3) Circle the word that correctly completes the sentence describing the conventions for intracellular (or whole-
cell patch clamp) voltage-clamp recording:

a. The voltage outside the cell is (zero / variable) mV (i.e., outside the cell is “ground”).

b. The current sensing electrode is the one (outside / inside) the cell.

c. Current is defined as the direction of flow of (positive / negative) ions.

d. Inward current, which flows toward sensing electrode, is recorded as (positive / negative).

e. Outward current, flowing away from sensing electrode, is recorded as (positive / negative).

f. Cl- flowing into the cell is considered an (inward / outward) current.

4) Answer the following questions based on recording conditions given:

a. In intracellular recordings under current clamp, when current is removed from the cell (i.e., a
“negative” current is applied) the result is a (positive or depolarizing / negative or hyperpolarizing)
deflection of the voltage trace.

b. In intracellular recordings under voltage clamp, a shift in holding voltage to a more depolarized
potential opens some cation-selective voltage-gated channels. The equilibrium potential for these channels is
more depolarized than the new holding potential. The current flow in response is (inward / outward) and the
deflection you observe is (positive / negative).

c. Again, while recording intracellularly under voltage clamp, you perfuse on a ligand for a
neurotransmitter receptor, opening new anion-selective ion channels. If your holding voltage is more
depolarized than the equilibrium potential for these ions, will the current response deflection be positive or
negative? Is the current response inward or outward?

d. You move your recording electrode outside the cell to make “field” recordings. After bath perfusion
of an unknown compound, you observe a positive deflection in your recording. This indicates that neurons in
the vicinity of your electrode (depolarized / hyperpolarized) in response to the compound.

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NRSC500 Problem set September 2017

5)
OUT IN

Na+ Na+

K+ K+

Cl- Cl- , A-

CELL MEMBRANE (selectively permeable)

OUT IN

Na 140 mM 8 mM
K 5 mM 130 mM
Cl 145 mM 14.5 mM

We are recording the voltage difference across the neuronal membrane shown above, where the ground
electrode (V = 0) is outside the cell, and Vm measured inside the cell (resting membrane, or equilibrium,
potential) is -70mV. Show steps of calculations on the back side of this page or a separate sheet.

a) Calculate ENa, EK, and ECl.

b) Cl- is replaced with the impermeant anion gluconate. The Vm is now -73 mV.
What is P Na / P K ?

Hint: Use Goldman-Hodgkin-Katz equation, and multiply through top and bottom by 1 / PK.

Under these conditions, the P Na / P K suddenly changes to 25. In the first “jiffy” following this sudden
change in permeability ratio, which direction does the current flow (outward or inward)? What is the
Vm at steady-state at this new ratio?

c) Cl- is restored to the bathing solution (and gluconate removed), bringing the Vm back to -67mV. A
drug is added to the bathing solution, resulting in increased Cl- conductance (i.e., the number of Cl-
permeable channels increases). Will the Vm (equilibrium potential) become more or less negative?

d) We are now recording under voltage-clamp:

i) Give the equation which relates IK to VH (the holding potential). Do the same for INa and ICl.
ii) For VH = -100 mV, which direction is the K + current flow (inward or outward)? Which direction
is the Cl- current flow?
iii) Sketch the I-V curve for a membrane containing only outwardly rectifying K+-selective channels.

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NRSC500 Problem set September 2017

6) The Hodgkin-Huxley equations predict the current response to rapid changes in membrane voltage, by
modeling the gating of voltage-dependent Na+ and delayed rectifier K+ channels. The following questions are
based on your lecture notes on the H-H equations.

a) The above plot (left panel, labeled “Na+”) shows the peak current-voltage (I-V) relationship for voltage-
dependent Na+ channels, and the corresponding conductance vs voltage plot (right panel).
i. For what range of test potentials is the Na+ channel I-V curve ohmic (linear)?
ii. How does the Na conductance plot predict the ohmic region of the peak I-V curve?
iii. Briefly explain why the I-V curve shows negative slope conductance from -50mV to -25mV.

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NRSC500 Problem set September 2017

6b) The plot below shows Na+ channel conductance vs. time in response to the test potentials indicated
(starting from -100 mV holding potential); note that the test potential is held for 10ms.

i. Superimposed on these traces, roughly sketch the K+ channel conductance vs time; amplitude is
relative (Hint: you may need to extend the time axis; these plots can be found in your lecture notes!)
ii. Based on these plots of Na and K channel conductance vs time for -2mV and +44mV (assuming
you are starting from -100mV where channels are in the closed but fully activatable state), sketch the current
responses to a sudden jump to a test potential of -2mV and a second sketch of a jump to +44mV, plotting
current vs. time, for both Na+ and K+ channels. You will need to take into account the driving force for
each current (assume ENa is +55mV and EK is -85mV).

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NRSC500 Problem set September 2017

7) A neuron has RA = 75 Ohm-cm, RM = 225 Ohm-cm2; the radius of the dendrite is 0.0002 cm.

a) If the dendritic membrane is depolarized by 60 mV at a distance of 0.01 cm from the axon hillock,
what will the amplitude of the depolarization be when it reaches the axon hillock? (Use the cable
equation – i.e., for calculating decay of voltage with distance; to simplify the problem, assume that
the cell body is the same shape – a cylinder – and same radius as the dendrite).

b) If the resting membrane potential is - 75 mV, will this depolarization be likely to result in the firing
of an action potential? (Assume threshold for firing is Vm = -40 mV).

c) You are recording from this neuron under voltage clamp at -70mV. After perfusing a hormone,
you notice an increase in the amount of positive current required to hold the cell at -70mV and a
decrease in the Rm. EK is -85mV, ECl is -60mV, and ENa is +55mV.

i. Of the ion channels most relevant to resting current (K, Cl, Na), which one was most likely
affected by the hormone? Did the hormone increase or decrease the open probability of these
channels? Give your reasoning.

ii. Assuming there was no effect on membrane capacitance, what effect did the hormone have
on the membrane time constant?

iii. What effect did the hormone have on the membrane length constant? Does this effect make
it more or less likely that a given current stimulus applied to a distal dendrite will trigger an
action potential at the soma?

iv. After washing out the first hormone, you test a second neuromodulatory agent. You again
find an increase in the amount of positive current required to hold the cell at -70mV but this
time the Rm is increased. Which ion channel(s) could be affected by this agent? Did the agent
increase or decrease the channel’s open probability? Give two ways you could determine
which class of ion channel was affected.

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NRSC500 Problem set September 2017

8)

Methods:
Patch electrodes contained (in mM): 130 K-gluconate, 10 NaCl, 2 Na2ATP, 0.3 NaGTP, 10 HEPES, and
0.6 EGTA, buffered to pH 7.4 and ~275 mOsm. Slices and outside-out patches were perfused with media of
the following composition (in mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1 CaCl2, 3 MgSO4,
0.2 CdCl2, and 10 glucose and tetrodotoxin (TTX, 0.5-1 µM) saturated with 95% O2 and 5% CO2, pH 7.4.
In A, the current recorded after jumping to test potentials ranging from -60 to +70 mV (10 mV increments)
from a pre-pulse holding potential of -30 mV was subtracted from the total current recorded after jumping to
test potentials of -60 to +70 mV (10 mV increments) from a pre-pulse holding potential of -110 mV, and the
difference current at each test potential is plotted.

The figure and methods section above are from a voltage clamp study on a K current. Please do the following:

1) Plot the activation and inactivation curves for the currents.

2) What is the maximum conductance of this current?
3) Estimate the ½ inactivation voltage for the current.
4) Describe what type of K current this is.
5) How would you prove that this is the current that you think it is? (e.g. What is the definitive blocker of
this channel?)