Neurophysiology – lecture 18

March 17, 2011

1 Review:
1. We have previously laid out the mathematical model developed by Hodgkin and Huxley to describe
action potential generation.
2. During Tuesdays lecture we showed that the model could explain a variety of phenomena associated
with A.P. production.
These explanations were available to us because the mathematical model allows us to read the numeric
values of all the important variables in the model: gNa , gK , INa , IK , IL , m, n, and h.
3. Explaining Threshold
(a) There was a sharp demarcation between suprathreshold and subthreshold; a stimulus of 7.67 did
not produce an A.P., a stimulus of 7.68 did elicit an A.P.
(b) By studying the screen presenting INa and IK data we saw that the absolute value of INa exceeds
the absolute value of IK just before the A.P. occurs. This means that I Net will be inward and
will be adding + charge to the inside of the membrane.
(c) However, INa depends on gNa , Vm , ENa , m, and h and IK depends on gK , Vm , EK , and n.
(d) So many factors influence the Threshold for A.P. production. Threshold is not attributable to
one factor.
4. Explaining Accommodation
(a) A.P.s are less readily elicited when the depolarization is produced by a ramp of current than when
it is produced by an abrupt increase in current.
(b) This is because h decreases slowly as the membrane becomes more depolarized. A slow ramp
allows time for this decrease in h to occur which then decreases gNa and hence INa .
5. Explaining Refractoriness
(a) After eliciting a first A.P. and waiting for a few milliseconds a second A.P. can be elicited only if
a larger stimulus is employed.
(b) It was seen that 8ms after the first A.P.
i. n is increased above resting levels which means that gK is larger and IK is larger making it
harder to generate an inward Inet .
ii. h is decreased which means that gNa is smaller and INa is smaller making it harder to generate
an inward Inet .
(c) Thus 2 factors are important in producing refractoriness.

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 6. Explaining Anodal Break
(a) Anodal Break occurs when a long period of membrane hyperpolarization is followed by the elici-
tation of an A.P. just after the hyperpolarization ceases.
(b) During the period of hyperpolarization h increases from 0.65 or so to about 0.85. This means
that when the hyperpolarization stops gNa is larger than usual and hence INa is also larger than
usual producing an inward Inet .
7. The Hodgkin - Huxley model is viewed as a “good” model because it describes all the A.P. properties
it was designed to describe.
8. But , in fact the model is better than that because it also describes:
(a) The conduction velocity of squid axon A.P.s observed to be 21.2 m/s and calculated by the model
to be 18.8m/s.
(b) The effect of tetrodotoxin shown in Figure 10C.
(c) The effect of tetraethyl ammonium shown in Figure 10C.
(d) The form and properties of the A.P. produced in the myelinated axons of the frog.
(e) etc.
Thus the Hodgkin - Huxley model is a “great” model in that it accurately describes many other
phenomena that it was not specifically designed to describe.
9. Note that the development and application of the model of axon A.P. production is an excellent example
of how science progresses.
(a) First a set of data were developed using the voltage clamp to produce families of INa and IK
curves.
(b) Then a mathematical model was developed to describe all these curves and it was observed that
the curves could be well described by the equations.
(c) Then the model was used to describe data like changes in Vm that occur during A.P.s and the
properties of A.P.s.
(d) Now we talk about activation and inactivation and other factors as the way to explain the processes
involved in A.P. generation, though what we are really talking about are the processes that are
part of the mathematical model.

2 Relating the variables in the H-H model to specific biological

mechanisms
1. One would imagine there are specific molecular mechanisms which correspond to the factors m, n, h.
2. First we note that when recording single channel currents using a patch clamp (Figure 12A ) the
opening and closing of these Na+ channels is not predictable.
Further, the presence or absence of a previous channel opening does not appear to predict the proba-
bility of another channel opening (i.e., channels dont have any short term memory). Behavior of this
type is described as “stochastic” and can only be described in terms of probabilities – the probability
of being open, Po .

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 3. In Figure 12A we also see that the summation of many (in this case 300) records in which the Na+
channel opened some of the time produces a current record like that under “2” in the figure.
4. This summed current record is very similar to the current INa recorded from entire segment of squid
axon when recorded during voltage clamping. So we assume that when voltage clamping we are
recording the current simultaneously obtained from thousands and thousands of Na+ channels all
stimulated at the same time.
5. Mathematically, this is to say INa = n Po iNa , where n = the number of channels available in the
membrane being recorded from.
6. Back in the first few weeks of class we noted that in the S4 transmembrane spanning alpha helix of
Na+ channels there was a Lys+ or Arg+ amino acid residue placed every third amino acid in the helix
(Figure 4A). Mutating these charged amino acids to neutral amino acids caused the Na+ channel to
lose some of its sensitivity to changes in transmembrane potential. So this S4 alpha helix was and is
presumed to contain the channels “voltage sensor.”
7. Voltage sensing is a property of both Na+ and K+ channels and is most clearly incorporated into the
H-H model by the parameters m and n which are highly voltage dependent (Figure 11B). Therefore,
we might expect that the properties of m might be explained by the properties of the S4 alpha helix.
8. The S4 alpha helix extends from one surface of the membrane to the other and thus is in a position
where it is exposed to the entire transmembrane potential. Now to us a -60mV resting potential doesnt
sound like much, but -60mV imposed across a membrane only 5nm thick is equivalent to a voltage
gradient of -120,000V/cm, so this is a very steep voltage gradient.
9. The fact that the intracellular voltage is -60mV relative to the extracellular surface means that the +
charges on the S4 alpha helix will be attracted in toward the interior surface of the membrane by the
resting potential.
10. When the intracellular potential is depolarized (made more positive) the attractive force on these +
charges in the S4 alpha helix will be reduced and then eliminated when the transmembrane potential
= 0mV. This reduction will allow the + charges on the S4 alpha helix to move outward away from the
interior surface of the membrane and of course the entire alpha helix will be moving at the same time.
11. The movement of these + charges on the S4 alpha helix is a form of current since by definition current
is the movement of + charge. This current is called the “gating current,” Ig .
12. This current would be defined as a capacitative current since no charges actually move from inside the
membrane to outside the membrane, rather the distribution of charge is altered by the voltage field.
Since the charge movement should be toward the outside of the membrane this should be recorded as
a current out.
13. If the movement of the + charges on the S4 alpha helix is responsible for opening the Na+ channels,
then we would also expect the gating current to occur prior to the INa .
14. At the top of Figure 12B we see currents elicited by step depolarizations in a voltage clamp recording
from a squid axon. The small outward current at the beginning of the current traces is where the
current we are interested in should be located but it is hardly visible among the much larger inward
current carried by Na+ .
15. To make gating current visible the preparation must be treated with TTX to block INa , be intracel-
lularly perfused with Cs+ to block IK and membrane capacitative, Ic , and leakage current, IL , must
be removed by adding current records after using equally large voltage steps in the hyperpolarizing
direction.
16. After all this is done the remaining current is Ig . Actually, Ig is so small that one must add the records
from 50 to 100 stimulations to get a figure like that shown in Figure 12 B bottom.

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17. The data do confirm that charges do move in the membrane when the membrane is depolarized and
that is movement must be instrumental in the opening of Na+ channels.
18. However, exactly what type of movement of S4 occurs is yet somewhat uncertain.
In Figure 12C the authors propose this is a movement straight upward toward the outside of the
membrane.
Others have proposed that the movement involves spiraling of the alpha helix moving the charges
upward but in a circular fashion.
More recently, based on X-ray crystallography data it has been proposed that this initial movement
produces a rather marked conformational change in the protein.
In sum there is no consensus about how the S4 helix moves or how that acts to open the Na+ channel
and hence how the movement relates to m.
19. Lastly, I briefly mentioned how inactivation is produced and hence how that might be related to h.
20. In Figure 12D we see a cartoon of a voltage-gated channel that inactivates. As shown on the right
hand figure, when the channel is open a portion of the channel molecule which is free to move in the
cytoplasm is thought to enter the intracellular mouth of the channel and block the passage of ions.
This is commonly referred to as the Ball and Chain mechanism to produce inactivation.
21. The evidence supporting this model is that:
(a) proteolytic enzymes (papain, pronase) when injected into the cell and allowed time to act eliminate
the inactivation process presumably by cleaving the “chain” linking the ball to the channel and
thus allowing the ball to float away.
(b) Using K+ channels called IA channels which inactivate strongly, experimenters have not only
eliminated the inactivation using proteolytic enzymes but then infused a synthetic peptide based
on the amino acid sequence present in the “ball” and saw that inactivation of the channel was
returned.
22. More recently it has been observed that inactivation by the ball is a stochastic process not one de-
termined by some molecular mechanism. This will mean that fitting its properties to the equations
describing h will be difficult.