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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
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
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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 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
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
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
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 =
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
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)
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.
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
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
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
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
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
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
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
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
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
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
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.
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