Neurophysiology – lecture 15

March 1, 2011

1 Review:
1. We enumerated several properties of action potentials (A.P.s) some of which were surprising; for ex-
ample that action potentials can be triggered by hyperpolarization and that they are not always
All-or-None.
2. One A.P. property we could explain is the fact that the peak of the A.P. goes to approximately +40mV,
instead of just 0mV.
Since the A.P. produces and is produced by a large (625 fold) increase in Na+ permeability, both the
G-H-K Equation and the Parallel Conductance Equation indicate that the Vm should closely approach
ENa which is generally near +50mV.
dV
3. However, most of the time during an A.P. does not equal 0, so the assumptions underlying the
dt
G-H-K Equation and Parallel Conductance Equation are not met and hence these equations will not
give correct answers.
dV
4. To circumvent that problem that does not equal 0 and that many variables in the equations we
dt
have been using are changing during the course of an A.P. neurophysiologists developed the Voltage
Clamp.
5. A voltage clamp allows the experimenter to set the transmembrane potential to some desired voltage
relative to ground and keep it at that potential. During the period of time that the voltage clamp is
“on,” the amount of current required to hold the Vm at the desired constant level is measured.
6. The voltage clamp establishes and maintains the Vm at the desired level by passing current through
the neuronal membrane across the input resistance of the neuron thus creating a voltage difference
between the inside and outside of the cell, I · Rin .
7. In the simplest experiment with a voltage clamp the Vm is stepped up from resting potential by +10mV
and a current is elicited which has a sharp initial outward current spike followed by a small steady
outward current. The spike is attributed to that current which is placing charge on the plates of the
membrane capacitor to create the step in Vm . The steady outward current is ionic current passing
through the membrane resistance thereby maintaining a change in transmembrane potential equal to
I · Rin .
8. If the Vm is stepped down from resting potential by -10mV a sharp initial inward current is followed
by a small steady inward current. As above, the initial current spike charges the membrane capacitor
and the steady inward current generates a change in Vm by passing current through the membrane
resistance.

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 9. Since the membrane resistance and capacitance are the same during these 2 voltage steps and the 2
voltage steps are in opposite directions, the 2 current records produced should be of equal magnitudes
but opposite directions. Hence, if these 2 current records are summed together the result should show
no current was present; thus the capacitative and leakage currents can be eliminated by making voltage
steps of equal amplitude but opposite directions and adding the resultant current records together.

2 The use of larger voltage steps

1. If the experimenter uses voltage steps that are larger than +10 mV, the current record obtained
becomes much more complex. For example, look at the top 5 current records portrayed in Figure 10A.
Looking at the voltage step to -35mV we see first a small capacitative spike followed by an inward
(down) current lasting for 4 ms and then a long lasting outward (up) current.
2. This total pattern of currents is assumed to be due to: IT = IK +INa +IL +Ic where ICl is replaced by IL
which represents not only Cl− but also other currents whose conductance through the membrane does
not change with changes in Vm . Note that this summation of 4 currents is a mathematical statement
of the “Independence Principle,” since the value of any one of these 4 currents can change without
changing the value of any other current in the summation. It also fits the Electrical Membrane Model
in that 4 separate current pathways are present in the model to allow the separate passage of the 4
different types of currents.
3. Given any one of these IT records, say that produced when the Vm was stepped to -35mV (a 30 mV
step from -65mV), the IL + Ic can be removed from it by recording the currents produced by a -30mV
voltage step to -95mV and adding these currents (IL + Ic ) to the record of the currents produced by
a +30mV voltage step. After this addition the I record will contain only IK and INa .
4. This “subtraction” procedure applied to records obtained with a variety of different voltage steps will
generate an entire “family” of current records similar to those seen in Figure 10A but which only
contain IK and INa .
In these “subtracted.”current records one should note that the earliest current rises rapidly and is
inward while the late current is outward and does not decay as long as the voltage step is maintained.

3 Dissecting this “subtracted” IT into IK and INa .

1. For extra credit I asked the class “For which of the current records in Figure 10A can you determine
the ion that carries it?”
2. Wen Xu jumped on the correct answer: “that current record generated by a voltage step to +52mV.”
Since +52mV equals ENa and at ENa , INa = 0, it must be that the “subtracted” current record obtained
from the voltage step to +52mV must contain only IK .
3. Of course knowing that the voltage step to +52mV produces only IK , is of little help since transmem-
brane potentials never reach that level of depolarization naturally. So how can one generate a pure IK
record at some other transmembrane potential, say 0mV.
4. Sean Moore correctly suggested you could alter the concentration of Na+ in the external medium
such that the extracellular concentration of Na+ equaled the intracellular concentration, 10mM. Then
10mM/10mM =1 and the log of 1 = 0, so ENa = 0 mV according to the Nernst Equation.
Then a +65mV voltage step to 0mV followed by a -65mV voltage step to -130mV would yield 2 current
records which when added together would produce a “subtracted” IT which only contained IK .

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 5. This general procedure could be repeated with a variety of different extracellular Na+ concentrations
and voltage steps thereby generating a family of IK records obtained at a variety of different trans-
membrane potentials. This family of IK records would look like Figure 10C bottom left.
6. Figure 10B shows the method to produce a current record which contains only INa .
If by altering extracellular Na+ we set the ENa equal to some Vm (in the figure it is -9mV) and then
do a voltage step to -9mV, the resultant current record will be an IK only record (curve B). If we then
do the same voltage step when the extracellular medium is regular seawater, the current record will
contain both IK and INa . Therefore, subtracting the IK record from the IK and INa record will leave
a record containing only INa (curve C).
7. Note that curve A and curve B meet after 5ms, so all the outward current recorded after this is IK .
8. Note that INa is always inward as one would suspect from its concentration gradient.
Note that INa increases in magnitude much more rapidly than IK .
Also note that INa increases in magnitude and then decreases in magnitude despite the fact that the
voltage step stays on.
9. Using a variety of extracellular Na+ concentrations coupled with the appropriate voltage step needed
to set Vm equal to the ENa established at the particular Na+ concentration, a family of INa records
can be obtained. This family would look like the family of current portrayed in Figure 10C bottom
right.

4 Use of channel blockers

1. Hodgkin and Huxley devised the method outlined above to isolate IK and INa from other currents and
from themselves and portrayed the results.
2. Subsequently, it was discovered that the addition of certain chemical compounds can also isolate INa
from IK and IK from INa . These compounds act by blocking the flow of current through one or another
ion specific channels in neurons.
3. For instance, tetrodotoxin (TTX) found in the puffer fish blocks inward Na+ currents through voltage-
dependent Na+ channels. Thus neurons and axons treated with TTX and studied by means of voltage
clamping display only K+ currents (Figure C bottom left).
4. Conversely, neurons and axons treated with tetraethylammonium (TEA) display only inward Na+
currents, because TEA blocks K+ channels.
5. Thus, using these pharmacological agents IK and INa can be determined rather more easily than was
the case in the studies of Hodgkin and Huxley.

5 Converting currents to conductances

1. As a final step in the process of transforming complex raw current records into a form that can be used
in generating action potentials and predicting the behavior of neurons, we need to convert the IK and
INa records we have obtained into gK records and gNa records respectively.
2. This can be simply done using the equations we have previously obtained:
INa IK
gNa = and gK =
Vm − ENa Vm − EK

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 Of course the current obtained at a given transmembrane potential (Vm ) and time must be used in
these equations with that potential present in the driving force function.
3. This procedure was used to generate the graphs of conductance vs. time shown in Figure 10D.
4. Note that while INa is inward and therefore has a minus sign, gNa is positive and in fact conductances
are scalar quantities and can only be positive. gNa is positive because (Vm − ENa ) is also a negative
number since ENa is a larger number than Vm , for values below the peak of the A.P.
5. Note that gNa rises more rapidly than gK , but that gNa also decreases despite the voltage step being
maintained. This decrease is attributed to a process called “inactivation” about which we will have
more to say later.
6. Also note that the rate of the rise of both gNa and gK increases as the amplitude of the voltage step
increases. Thus both gNa and gK are voltage dependent.