Ionic Basis of the Action Potential

I hope you’re feeling comfortable with the principles that govern the formation of the resting

potential, because those same principles apply to the action potential, which is the mechanism that

the nervous system uses for long distance signalling. There is, of course, a difference between the

tow and that is that during the action potential, the membrane isn’t resting--its potential increases

and decreases very rapidly because the membrane actively changes its permeability to different ions.

What I’m going to do is first tell you about the history of ideas to explain the action potential,

describe a series of experiments conducted by Alan Hodgkin and Andrew Huxley around 1950 that

established the modern explanation for the AP, then discuss what cell and molecular biologists have

been able to add to their story in the last 40 years.

First, the history. As I previously mentioned, Galvani suggested in 1791 that animals used

electrical signals in their nervous system, and this was established conclusively in the mid-1800s by

the German/French scientist Dubois-Raymond. However, it wasn’t until the early years of the 20th

century that anyone tried to suggest a mechanism for how these signals could be generated, and the

first person to do that was Julius Bernstein, whom we’ve already discussed. Bernstein had applied

the Nernst equation to the study of the resting potential and concluded that the membrane of nerve

cells was selectively permeable only to K+ at rest (as we now know, this isn’t exactly true). He

knew that the interior of nerve cells was negative with respect to the outside of the cell in resting

cells. And he made an effort to measure the change in the membrane potential during an action

potential, which normally lasts only a few thousandths of a second. What he was able to show was

that the membrane potential became much more positive during the action potential. Bernstein in

fact thought that the membrane potential changed from -60 mV or so to 0 mV for a brief time

during the AP; i.e., the membrane potential became 60 mV more positive than normal. His

explanation for this was that a resting membrane was selective permeable to K+, but when the cell

was “excited”, somehow the membrane became so disorganized that it would allow any ion to pass

through it freely; i.e., the permeability for all ions was essentially infinite for a brief period. What

effect would infinte permeability to all ions have on Vm? He published his idea in 1902 and it was

the standard explanation of the action potential for 40 years.

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 Once again Bernstein was partly right and partly wrong. In fact in the same issue of the same

journal in which Bernstein published his explanation of the action potential, another scientist named

William Overton published a paper that contradicted Bernstein’s basic hypothesis. Overton

showed that if he removed Na+ ions from the liquid in which he immersed a frog muscle, he

abolished the ability of the muscle to conduct action potentials. Why does this contradict

Bernstein’s hypothesis? Because if Bernstein were right, even with no external Na+, when the

membrane permeability went to infinity, Na+in, K+ and Cl- would rapidly cross the membrane and

cause Vm to go to 0. In other words, in Bernstein’s model the action potential shouldn’t depend on

the external concentration of any ion. Overton thought that his results implied that the membrane

maintained its ability to select among ions during an action potential, but that the permeability for

Na+ rose dramatically. However, he couldn’t think of an experiment to test whether that was true,

and so his idea was passed over and ignored in favor of Bernstein’s.

That situation persisted until the 1930’s when two important technical advances allowed scientists

to measure the membrane potential at rest and during the action potential much more precisely than

ever before. The first advance was the rediscovery by J.Z. Young, a British physiologist, of the

squid giant axon, a relatively enormous nerve cell. With this axon it was possible to slip a thin

copper wire inside it and use the wire as an intracellular recording electrode. By comparing the

internal charge, as measured by this electrode, with a reference electrode outside the cells, it was

possible to get a much more accurate reading of Vm and membrane resistance (which is related to

the permeability of the membrane to ions) than ever before.

The first experiments, conducted by the Americans Cole and Curtis, showed that during an action

potential the permeability of the membrane increased but did not become infinite as proposed by

Bernstein. Moreover, Cole and Curtis and the Britons Hodgkin and Huxley soon showed that

during the peak of the action potential (AP) the membrane potential was actually more positive

inside than out, rather than being equal to zero, as predicted by Bernstein. What does this result

indicate about membrane permeability to ions during the action potential? It means that the

membrane is not infinitely permeable to all ions, but must remain selectively permeable to ions

during an AP. At this time, WWII began and these scientists took up war-related research.

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 After the war, Hodgkin and Katz returned to consider this problem and they basically came up the

same idea that Overton had proposed--that the permeability of nerve cells increased greatly to Na+

during an action potential. They first showed theoretically that this would cause a big increase in

the resting potential, and then they did some experiments to suggest that external [Na+] had a large

effect on the size of the AP. Their theoretical explanation was based on the GHK equation that we

encountered in our discussion of resting potential.

Remember: Vm = RT ln PK[K+]out + PNa [Na+]out For mammalian muscle, PNa/PK = 0.02; K+in =
F PK[K+]in + PNa [Na+]in 4 mM; K+out= 140 mM, Na+out = 142 mM;
Na+ in = 12 mM ; ENa = +62mV; EK = -90 mV

When b = 0.02, Vm = -76 if you plug all those values into the GHK equation. Hodgkin & Katz

said, suppose we could increase PNa a lot all of a sudden, and make it 500 times bigger than usual.

What would happen to Vm, according to our equation? Work it out for yourself; it’s something

you might be asked to do on an exam. What would Vm be? (Answer: +43 mV). In other words

a big increase PNa would cause the membrane potential to move away from EK and close to ENa. So

Hodgkin & Katz reasoned that the peak of the action potential (draw what the “peak” is) should

vary according to [Na+]out. They thought that if the peak amplitude was near ENa, then they could

vary ENa by changing [Na+]out. i.e., if Vm (AP) ~ ENa = RT/F ln [Na+]out/[Na+]in = 58 log [Na+]out -

58 log [Na+]in, then varying [Na+]out should cause the maximum Vm of the AP (action potential) to

change. To test this, they varied [Na+]out and measured the maximum amplitude of the AP. What

else should they have tested as a control for their hypothesis?

See Figure 2.8 in Purves et al., Neuroscience

They got a nearly straight line of positive slope when they plotted Vm (AP) vs log [Na+]out, but no

change when they varied [K+]out or [Cl-]out. So they proposed that the Action Potenial could be

caused by a sudden, but short-lived increase in the PNA of the membrane. If their hypothesis was

correct, then when PNa increased, Vm would become positive (a depolarization) and when PNa

decreased, the Vm would return to its normal negative value (a repolarization). This is the sodium

theory of Hodgkin & Katz, published in 1949. In the following years Hodgkin, working with

Huxley, figured out a way to test this hypothesis. I want to describe the conclusions they reached

first, then after that I’ll explain the experiments on which they based their conclusions.

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 As I indicated above, the Action Potential revealed by the work of Cole and Curtis and

Hodgkin and Huxley in the 1930s had several features (see Box B in chapter 2 of Purves et al.)

Various phases of the AP included resting potential, the rising phase, threshold, the overshoot, the

falling phase, and the undershoot. In addition, it was either impossible or difficult to stimulate a

neuron to fire a second action potential in the period immediately after one had occurred, and these

were referred to as the absolute and relative refractory periods. They noticed that once a cell’s

membrane potential passed the threshold value, the AP was stereotyped. That is, it always had the

same amplitude and time course for a given cell. But as long as Vm remained below the threshold,

no AP occurred; that is, the AP was all or none, depending on whether the Vm exceeded threshold.

The triumph of Hodgkin and Huxley’s work in the late 40s and early 50s is that they provided an

explanation for all these features of the AP, and they provided compelling experimental evidence

that their explanation was right.

Well, we now think that Hodgkin and Katz’s sodium theory was correct; that is, that the

membrane potential changes during an action potential because the membrane permeability to ions,

especially sodium ions, changes. I’ve already given you a mathematical argument, based on the

GHK equation, that changes in membrane permeability should cause changes in membrane

potential. Hodgkin and Huxley showed that this was exactly true--they measured membrane

potential and membrane permeability throughout the course of an action potential and showed that

the changes in ionic permeability of the membrane could account for changes in the membrane

potential.

Actually, what they measured was not membrane permeability, but membrane conductance,

which is a much easier value to determine experimentally. They used something called the parallel

conductance model, rather than the GHK equation, because it's easier to compute changes in Vm as a

function of changes in conductance. The idea of this model is that anytime the membrane potential

is constant, the rate of change of Vm = 0, which is true only if there’s no net movement of ions

through the membrane: Im = 0 = IK + INa (ignoring ICl). And Ohm’s law says V = IR or I = gV.

(Conductance, symbolized by g, is defined as 1/R). Since V for an ion equals the difference

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between the Vm and the equilibrium potential for the ion, Iion = gion(Vm-Eion). So IK + INa = 0 =

gK(Vm-EK) + gNa (Vm - ENa).

Rearranging gives Vm = gNAENa + gK EK = gNa/gKENa + EK

GNa + gK 1 +gNa/gK when gNa/gK changes, so does Vm.

This is just another way of saying that if something changed gNa, it would cause a change in

Vm. What Hodgkin &Huxley showed is that the agent that changes gNa is Vm itself. That is, an

increase in Vm causes an increase in gNa, which in turn causes a further increase in Vm. This is a

self-reinforcing action, or positive feedback--a similar phenomenon is seen in the relationship

between reaction rate and heat in gunpowder.

So if Vm increases gNa, then Vm will continue to increase until it reaches what value? (ENa).

That explains the overshoot, but the action potential, as you know, quickly returns to the resting

potential. Why? Because gNa returns to its original value through a process called inactivation. In

other words the increase in gNa is temporary. And this rise and fall in gNa would be enough by itself

to cause a rise and fall of Vm. But in addition, in the squid giant axon that Hodgkin & Huxley

studied, gK also changes; it increases during the AP, so that gNa/gK is actually smaller after the

Action Potential than before, making the Vm more negative than the normal resting potential (the

undershoot).

After the gNa is inactivated it takes a small, but finite, period of time for it to recover to the

resting value. During this period, gNa no longer changes its value when Vm increases. The

consequence is that no matter how much one increases Vm (“depolarizes” the cell), there is no

increase in gNa, and therefore no increased sodium entry, and therefore no AP. This period when

gNa is locked into a non-conducting state is called the absolute refractory period. However, if Vm

stays at or below the resting membrane potential for a few milliseconds, gNa reactivates (unlocks)

and the cell returns to its original level of excitability.

Because gK stays high for a while after the AP, even while gNa is returning to normal, the

resting potential is less than normal, meaning that Vm is more negative and therefore farther from

threshold than usual. During this period, it is harder than usual to bring the cell to threshold (a

bigger change in Vm is necessary), and this is called the relative refractory period.

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 I've given you Hodgkin &Huxley’s explanation for refractory periods, undershoot and

overshoot, and now I want to tackle the tricky notion of why there is a threshold, which is defined as

the value of Vm below which there is never an AP and above which there always is. The cell acts

discontinuously; one doesn’t see a fraction of an action potential. How can this discontinuous

behavior arise from continuous variation in the sodium and potassium conductances? It turns out

that at the threshold, the influx of sodium (the sodium current into the cell) is exactly balanced by

the efflux of potassium (the potassium current out of the cell). When else are those two currents

balanced? (At the resting potential). How can it be that the two currents balance at two different

values of the resting potential. I’m going to give a semi-quantitave argument for how this might

work. Remember that whenever the rate of change of Vm = 0, then Im = 0 = IK + INa or INa = - IK; or

gNa(Vm - ENa) = -gK(Vm-EK). At the resting potential gNa is small, Vm-ENa is large, gK is large and Vm-

EK is small; the products of those two large and small values are equal. If the membrane potential is

depolarized, then I’ve said that this causes gNa to get larger. That means that INa (the product of

gNa(Vm - ENa)) will increase; more sodium ions will be entering the cell per given unit of time. But

as Vm increases the value of Vm - EK will get also larger, even though gK stays relatively constant.

That means that IK (= gK(Vm-EK)) will also increase when Vm increases, and more K+ will exit the

cell per unit time. The threshold is the point at which the increase in sodium inflow is exactly

balanced by the increase in potassium outflow. Below the threshold, the increase in K+ outflow is

bigger than the increase in sodium influx, and more positive charge leaves the cell than enters,

causing Vm to return to rest. Small depolarizations (below threshold) don’t cause enough of an

increase in sodium conductance to cause an AP. At or above threshold, the increased rate of

sodium entry exceeds the increased rate at which potassium leaves, and therefore the membrane

potential starts to increase (as Vm moves toward ENa); this in turn causes a further increase in sodium

conductance, more rapid movement of Na+ into the cell, a bigger change in Vm and so forth, causing

the rapid depolarization of the action potential that lasts until the sodium conductance inactivates.

In summary, the Hodgkin-Huxley explanation is that the AP is caused by a rapid increase in

gNa, that allows Na+ to rush into the cell along its electrochemical gradient, followed by a rapid

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decrease in gNa (inactivation) and a slower increase in gK. These latter two events bring the Vm back

below zero to near the normal resting potential.

Now Hodgkin and Huxley suspected that the description I just gave you was correct, but

they needed a way to test this idea experimentally. To do such a test, they’d have to measure gK, gNa

and Vm constantly during an AP, a prodigious task they accomplished in 1950 and published in

1952 and 1953. It was a technical and intellectual tour de force because:

a). They thought that gNa = f (Vm, t) and gK = f'(Vm, t). So there were 4 variables, Vm, gNa,

gK, and t; and 3 of them interact with and affect each other—Vm, gNa, and gK.

b. The changes occur very rapidly during the initial phase of the AP: ∆Vm/∆t ≥ 700

Volt/sec, making it hard to do accurate measurements within short periods of time.

c. There is a capacitive current that complicates measurement of ionic currents--essentially

the problem you encountered with the “stimulus artifact".

Thus the natural situation is too complex to study as a whole. Hodgkin &Huxley decided

to simplify the situation by eliminating one of the variables, ∆V/∆t--i.e., they devised a method to

hold Vm constant while measuring gNa, gK and t.

To do this they used an apparatus called a voltage clamp that was first designed by K.S.

Cole. This is an electronic machine that can shift Vm instantly (within microseconds) and hold it

constant at some new value. (See Box A, Chapter 3 of Purves et al., Neuroscience.)

Using this system they could isolate a large segment of membrane and hold it isopotential

so that the action potential occurred throughout the whole piece of membrane at once. The voltage

clamp works as follows: Starting at the existing membrane potential of the axon (the “holding

potential”), you dial in the desired Vm on the voltage clamp. The voltage clamp passes current

through the positive electrode until the voltage measuring electrodes indicate that Vm = Vcommand

(the voltage that you chose and dialed into the voltage clamp). But if you increase Vm, this causes

an increase in gNa of the membrane, which means that there is now an increased likelihood that Na+

ions will enter the axon, which will tend to make Vm more positive. However the slight increase in

Vm is detected by the Vm-sensing input to the voltage clamp, which now passes current in order to

maintain Vm at the command potential. In this case, it will pass negative current in order to

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counteract the accumulation of Na+ inside the cell. In other words, the electronic feedback circuit

removes positive charge and adds negative charge just as fast as Na+ enters, keeping Vm constant.

Later gNa declines, Na+ stops entering, but then gK increases and K+ starts flowing out of the cell

more rapidly, causing Vm to become more negative. Again the machine senses this and passes

positive charge to counteract the efflux (outflow) of K+ and to hold Vm constant. While all this is

going on, the current being passed by the voltage clamp--either positive or negative--is being

recorded by a recording device (essentially a fancy current meter). If everything works, then ∆Vm

= 0, that is the membrane potential is constant, so there must be no net current through the

membrane (according to Ohm’s law): Imembrane = 0 = IK + INa + Iother ions + Ivoltage clamp, or

-Ivoltage clamp = IK + INa + Iothers.

Purves et all show slightly cleaned up versions of the results that H&H obtained. The first one (Fig.

3.1B in Purves) shows their measurement of the total current crossing the membrane (= I voltage clamp)

when the membrane of the squid axon was set to different voltages. Initially there is an inward rush

of positive charge (or current) followed by a later outflow of positive charge that lasted as long as

the membrane was kept at the depolarized voltage. (At very high values of Vm, there is no inward

current; it’s all outward. Why?)

While these measurements give the total movement of ion across the membrane (= -I voltage

), they don’t tell us whether the inward positive current is because Na+ is entering the cell, K+
clamp

entering the cell or some other positive ion is entering the cell (or Cl- leaving). So Hodgkin &

Huxley had to find a way to separate out the sodium and potassium currents, which they did by

removing extracellular Na+ and replacing it with an impermeant positive ion, choline. Now when

they repeated their measurements, they saw a current that was essentially all caused by the

movement of K+ ions (Figure 3.4, Purves, middle trace ("Na-free")). If they then assumed that Itotal

= IK + INa (ignoring the other possible currents), they could also calculate INa, by simply subtracting

the IK measured in the absence of sodium from the total current measured in the presence of

sodium.

From these measurements of the time course of the sodium and potassium currents, they

could compute gNa and gK during the action potential, using the version of Ohm’s law that we

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discussed earlier: IK = gK (Vm - EK) and INa = gNa (Vm - ENa). Since Vm is held constant throughout

the experiment, and EK and ENa don’t change (the movement of a few ions across the membrane is a

drop in the bucket so the total concentrations of ions inside and outside don’t change), Vm-ENa and

Vm - EK are constants. So changes in I (current) are directly proportional to changes in g

(conductance, which is related to permeability).

In other words the principle of the voltage clamp is to measure the movement of ions

electronically and to calculate the changes in the membrane conductance to particular ions over time

using Ohm’s law. Then one changes Vm to some new value, and repeats the experiment. By doing

enough measurements of this sort, Hodgkin &Huxley could figure out how gNa and gK varied as

functions of Vm and time.

Fig. 3.6 in Purves et al. shows gK and gNa as continuous functions of time at several

different values of Vm. Note the differences between them. At low values of Vm, gNa increases and

remains high, but at higher values of Vm, gNa increases rapidly (more rapidly as Vm gets larger) and

then decreases quickly back to essentially zero, a process they called “inactivation”. gK increases

more slowly than gNa as a function of Vm, but once it reaches its maximum value, gK stays constant

as long as Vm is constant. That is, gK doesn’t inactivate (at least not in squid axons; there are some

neurons that have an inactivating gK).

Hodgkin & Huxley supposed that there were two opposing processes that affected gNa when

Vm was increased. A very fast process (activation) caused the gNa to increase greatly, while a slower

(but still rapid) process called inactivation eventually shut down gNa and caused a decrease. On the

other hand gK was subject only to an activation process that was relatively slow compared to what

was happening to gNa. H&H further showed that gNa and gK eventually returned to their original

values if they shifted Vm to some positive value for a while, then shifted it back to the original

resting potential. That is, just as gNa activated and inactivated when Vm was increased, it reactivated

when Vm was decreased again. Similarly, gK went up when Vm was increased and went back down

when Vm went down again; the inactivation and activation processes are reversible.

From their measurements of gNa and gK as functions of Vm and t, Hodgkin & Huxley

derived a series of empirical equations for gNa and gK as functions of Vm and used these to compute

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what would happen to Vm during an AP. They did this by changing t in 0.01 msec increments,

recomputing gNa and gK, figuring out how that would alter Vm, changing t again, seeing how the new

value of Vm would alter gNa and gK, and repeated that process over and over. Huxley did the

calculations on a manual desk calculator because computers and electronic calculators weren’t

available. It took him 3 weeks to compute all the values for one AP and mostly they put the wrong

parameters into their equation, and they got something that didn’t look anything like a real AP. But

the next slide shows one computed AP compared with a real AP (Fig. 3.8B and C in Purves et al.).

The computed AP depends on certain assumptions about the relationship between Vm and gNa and

gK that I've explained. As you can see, the computed action potential has an amplitude and time

course very similar to what happens during a real AP, providing strong support for their hypothesis

that alterations in membrane ionic conductances control changes in Vm during an AP. These

calculations allowed them to account for the size and shape of the AP, the increase in membrane

permeability during the AP, the absolute and relative refractory periods, the threshold and a variety

of other measured characteristics of the AP (I’ve already given you their explanations), which

indicated that they were on the right track with their explanation of the AP.

Now this calculation was based on an unrealistic circumstance of having the whole axon go

through an action potential simultaneously. In reality the action potential starts in one place and

travels along the axon. So they also computed what would happen during a conducted Action

Potential, as shown in the fourth slide (this one's not in the book). They found that Vm rises above

threshold before gNa and gK change at all. Why? That’s because electrical current is flowing along

inside the axon from an adjacent region and changing Vm, via a so-called local circuit, which we’ll

discuss next subsequently.

To reiterate the important points about action potentials that I’ve made so far:

1.Increasing Vm causes an increase in gNa, allowing Na+ ions to enter the cell rapidly; this

influx of a positively charged ion (an inward current) causes a further increase in Vm.

2. Eventually gNa inactivates, so gNa goes essentially to zero, and no more Na+ can enter the

cell.

3. Vm also increases gK, allowing K+ to exit the cell (an “outward current”).

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 4. The decrease in gNa caused by inactivation and the increase in gK brings Vm back to or

below (more negative than) its original value.

5. The return of V to a negative value allows gNa and gK to recover to their original

conditions.

6. A change in membrane potential at one place on the surface of a cell (such as during an

AP) can depolarize an adjacent region of the cell and cause it to fire an action potential.

Created by Gary Reiness

Last updated Jan. 26,2005

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