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Chapter 4
Hodgkin and Huxley and the Squid Giant Axon:
The Discovery of How Ions Generate Action Potentials
In the previous chapter, you were introduced to the action potential together with the ways
that the voltage gated ion Na
and K+ channels operate to generate it. But how did scientists
discover ion channels and how did they discover the types of gates that Na
and K
channels
have and the time scales upon which they operate? The answers came from the extraordinary
studies of Alan Hodgkin and Andrew Huxley, two of the greatest neuroscientists of the last
century. In 1952, Hodgkin and Huxley wrote a series of papers that described the experiments
they conducted that were aimed at determining the laws that govern the movement of ions in a
nerve cell during an action potential. These were among the greatest experiments ever conducted
in neuroscience and were the first to explain how ion channels work, which ions produced which
effects on the membrane potential, and provide the basis for our understanding of how action
potentials are generated and how they propagate down axons. Their insights form the bedrock of
modern neuroscience. For this achievement, they were awarded the Nobel Prize in Medicine or
Physiology in 1963.
The Hodgkin and Huxley experiments were conducted on a most unusual animal, the squid,
and used a newly developed electronic method called the voltage clamp. The reason they used
the squid for their experiments is because the squid has giant axons that are used for escape (Fig.
1). To give you an idea how large these axons are, a typical axon in your body has a diameter of
about 2 μm (1.0 μm is 1/1,000,000 of a meter) whereas the diameter of a giant axon is 800- 1000
μm, almost a full millimeter. In biology, bigger is always better because something larger is
easier to see and far easier to manipulate than a smaller version. The squid giant axon beautifully
illustrates the advantages of being large; indeed, it is so large that it can be seen with the naked
eye and it can be cut out of the squid and placed in a dish filled with seawater. Wires, to record
membrane potentials or to pass current, can easily be inserted down its interior, thereby allowing
Hodgkin and Huxley, for the first time in history, to record a resting potential. The wire also
allowed them to stimulate the axon and record the changes in membrane potential and ion
currents that occurred during an action potential. As we shall see, the features they observed and
manipulated were critical for understanding the ionic basis of the action potential, and could not
have been done on any mammal because the axons in mammals are much too small.
Hodgkin and Huxley used a new technique called voltage clamp to evaluate the ion currents
that generate an action potential. The rationale for using voltage clamp and how it is
accomplished is explained in a later section.
Fig. 1. Left panel shows a squid
and its nervous system. The giant
stellate ganglion is where the fibers
from the brain make synaptic
contacts with the giant axons. A
giant axon is shown in the right
panel. The giant axon is surrounded
by a large number of smaller axons
that travel along with it.
Ohm’s law relates voltage, current and conductance
Before continuing, I would like to explain four terms in a slightly more precise way than
was done previously. The terms are not new and were used before in a fairly lose way. They
are:
- Membrane potential, which, as mentioned above, is the degree of negativity (or
positivity) inside the cell relative to the outside and is measured in millivolts (mV).
- Current, which is nothing more than the flow of ions through the channels in the
membrane. The more ions that pass through channels, the larger the current. The flow of current
is referred to as “inward current “when positive ions flow into the cell and is referred to as
“outward current” when positive ions flow out of the cell. “I” is the symbol for current in
equations. Current is in units of Amperes and the small currents in neurons are usually
nonoamperes (nA) or less.
- Conductance is the ease with which an ion can pass through a channel. Conductance
means the same thing as permeability, but it can be measured, represented quantitatively, and is
used in equations, as shown below. The symbol for conductance is g. Thus g na
refers to sodium
conductance and g k
refers to potassium conductance.
- Driving force is the net force acting on an ion that drives it into or out of a cell. The
driving force on an ion is the difference between the equilibrium potential for the ion and the
membrane potential, (E ion
- V
m
The four terms are related and linked together by Ohms law. Ohm’s law states that the
amount of current flowing through a circuit is equal to the conductance times the driving force.
It is written as Current (I) =conductance (g) x driving force (E ion
- V
m
) or I=g x (E ion
- V
m
The relationship among the four terms is linear, straightforward and is illustrated diagrammatically
in Fig. 2.
potential. That is, they wanted to set the membrane potential at one voltage and then ask several
questions about the behavior of the conductances (the flow of Na+ and K+ through the
membrane) when the membrane potential was fixed at one value. One question was, “How do
the conductances for Na+ and K+, activated by one voltage (a particular, unvarying membrane
potential), behave over time?” Another question was, “How quickly are the conductances
activated and do they turn off over a period of milliseconds, or do they remain activated so long
as the membrane potential does not change?” There are several problems in making these
evaluations - all revolving about the fact that the action potential tends to get in the way because
everything is changing from moment to moment.
One problem that Hodgkin and Huxley knew before they began their experiments is that the
conductances for Na+ and K+ are not constant during the action potential. As Na+ and K+
conductances change, so does the membrane potential, and once the membrane potential
changes, conductances also change. In these circumstances, there is no way to evaluate whether
it is the membrane potential that is driving the conductance changes or whether the changes in
conductance drive the membrane potential. Said another way, there are three variables changing
at the same time and there is no independent variable - instead, we have three dependent
variables, time, conductance and voltage, all of which are changing together.
Another problem is that the action potential is conducted along an axon. This conduction
creates problems for their measurements, since they wanted to measure only what is happening
in a single small patch of membrane. Adjacent membrane regions will however greatly influence
their measurements, and it will be impossible to dissociate events in one patch from events in
adjacent patches.
Voltage Clamp to the Rescue!
The way in which Hodgkin and Huxley overcame these difficulties was to employ the
voltage clamp technique. This method has the advantage of holding the membrane potential
constant over the entire length of an axon while recording the currents that flow into and out of
the axon through ion channels. In other words, voltage can be held constant along the entire
axon while changes in current flows are recorded over time. The reason why this is so important
is that it allowed Hodgkin and Huxley to “freeze” the action potential along the length of the
axon and to record the changes in the Na+ and K+ currents at one membrane potential that did
not change in time.
How is it possible to hold the membrane potential constant, so that it does not change,
even when ion channels are opening and allowing positive charges to enter or leave the cell?
The diagram in Fig. 3 illustrates the voltage clamp circuitry. A voltage-sensing electrode (solid
black line in squid axon) is inserted down a length of a squid giant axon. This electrode is
connected to the oscilloscope that measures the membrane potential. The output of the
oscilloscope (which is the membrane potential (V m
)) is connected to one of the two inputs of a
differential amplifier (input A in Fig. 3). Input B of the differential amplifier is from a variable
voltage source (a dial in the diagram that allows the voltage to be set by the experimenter). The
differential amplifier is an amplifier that puts out a current (C current
in Fig. 3) that is proportional
to the difference in the voltages presented to the two inputs, A and B. In this way the
experimenter can set whatever voltage at B he desires. The current put out by the differential
amplifier, shown by the red line in Fig. 3, is fed to is an ammeter, which measures the current put
out by the differential amplifier, and then to a wire inserted down the length of the axon (dashed
red lines).
The voltage clamp circuitry operates in the following way. The experimenter sets the
voltage at B to some predetermined value by turning a dial. In this way the amplifier is given the
instruction: inject whatever current is necessary at C so that the membrane voltage becomes, and
is kept equal to, the voltage at B. The current flowing out of C (which is measured by the
ammeter) is equal to, but has the opposite charge of, the current flowing through ion channels
across the membrane. The current through C then counteracts any current flowing across the
membrane. For example, if positive charges leave the axon, the loss of positive charges make
the inside of the cell more negative. That negativity is immediately sensed by the differential
amplifier and the differential amplifier then injects an equal amount of positive charges back into
the axon. By replacing the charges flowing out of the axon in this case, the membrane potential
is held at a constant voltage that is equal to the voltage at input B. Once the current flowing
across the membrane is known (from the current measured by the ammeter), the membrane
conductance (g) is easily calculated from Ohm's law as g=I/V , where g= conductance of
membrane, I = current measured by the ammeter, and V is the voltage set by the experimenter.
Fig. 3. The main circuit elements required for voltage clamping. The squid giant axon is removed from
the animal and placed in a dish filled with seawater. Two wires are inserted down the entire length of the
axon. One, shown as a solid black line, measures the charges on the inside of the axon and is attached to
a wire that if fed to an oscilloscope. The other input to the oscilloscope is from a ground wire placed in
the seawater. Thus the oscilloscope measures the membrane potential (Vm), which is the difference in
charge between the inside and outside of the axon. The output of the oscilloscope, the Vm, is fed to the A
input of the differential amplifier. The voltage fed to the other input (B) of the differential amplifier is set
by the experimenter. The voltage he feeds to the B input is determined by turning the dial on the variable
voltage source. The differential amplifier puts out a current that is proportional to the difference in
voltage at its two inputs, A and B. That current put out by the differential amplifier is fed to an ammeter,
which measures the current, and then to the second wire in the axon (red wire). The current though the
red wire then changes the membrane potential along the entire length of the axon at exactly the same time
until the membrane potential, Vm, has the same value as the voltage set by the experimenter. At that
membrane potential, no further current flows into axon because both inputs to the differential amplifier
Sorting out the contribution of Na
and K
One way to sort out the contributions of the ions is by substitution experiments. For
example, we would suspect that the initial inward current is carried by Na
. To test this, we
could remove most of the Na
from the external bathing medium so that there is a higher
concentration of Na+ inside than outside of the cell, and E Na
is now - 10 mV (we could substitute
choline, a large positively charged molecule for NaCl in the preparation of the Ringer's. This
preserves the osmolarity and the total charge of the extracellular solution, but will block current
through the Na+ channel, as choline is too large to pass through the channel).
Fig. 4. Records of currents
recorded from squid giant
axon when the membrane
was stepped from rest (- 70
mV) to - 10 mV. The axon
in this experiment was
bathed in normal seawater.
The depolarization opened
ion channels that first
caused an inward current,
presumably carried by Na+,
which then shut off. The
inward current was
followed by an outward
current that was thought to
be carried by K+ ions.
We would expect that when the membrane is clamped to - 10 mV and when E Na
is - 10 mV, the
inward current should disappear, because when the Na+ channels open, the number of Na+ ions
Fig. 5. Records of currents recorded from
squid giant axon when the membrane was
stepped from rest (-70 mV) to - 10 mV. The
axon in the experiment shown in the top
panel was bathed in seawater in which the
Na+ concentration was severely reduced so
that E Na
was - 10 mV. The depolarization
opened Na+ channels but there was no
inward flow of Na+ because the membrane
potential was at the sodium equilibrium
potential, E NA
. After a short delay, there was
a prominent outward current carried by K+
ions as the delayed K+ channels opened. The
axon in the experiment shown in the bottom
panel was bathed in normal seawater that
also contained tetrodotoxin (TTX), a
powerful toxin that selectively blocks Na+ channels. Here too there was no inward current because the
Na+ channels that were opened by depolarization had their pores blocked by TTX and thus could not
conduct Na+ ions. These and other experiments proved that the early inward current is carried by Na+
ions.
driven out of the cell by its concentration force is exactly equal to the Na+ attracted back into the
cell by the electrical force. The only current that should remain is the outward current, since this
current is assumed to be carried by K+. This is exactly what is observed (top panel in Fig. 5).
Another way of showing that the initial inward current is carried only by Na+ ions is to
employ a toxin known as tetrodotoxin (abbreviated TTX). TTX is a neurotoxin isolated
primarily from the eggs and ovaries of the Japanese puffer fish. [While the fish is considered a
delicacy in Japan, the toxin is deadly, and only specially licensed chefs are allowed to prepare it.
Eating puffer fish is said to cause tingling sensations.] The action of TTX is to block the voltage
sensitive Na
channels. Thus, when TTX is used to treat a squid giant axon, the inward current
disappears, as shown in the middle panel of Fig. 5 and the top panel of Fig. 6
Proving that the delayed outward current is carried by K+ is more difficult. The
intracellular K+ concentration [K+]i is much greater than the extracellular K+ concentration
[K+]o and varying [K+]i is not feasible. However, K+ permeability is selectively blocked by
the drug, tetraethylammonium (TEA). Addition of TEA to the fluid bathing an axon under
voltage clamp results in the loss of the delayed outward current, leaving only the early inward
current. This is shown in Fig. 6 by the inward current (red) measured when K+ channels were
blocke by TEA. Indeed, Fig. 6 also shows that the total current measured during voltage clamp
is simply the linear addition of the inward current, carried by Na+, and the outward current,
carried by K+. The action potential is composed entirely of Na+ and K+ currents; nothing more
is required.
A model of membrane channels
Based on the graphs of Na+ and K+ conductances in Fig. 7, Hodgkin and Huxley
proposed the principal features of Na+ and K+ ion channels that produce the action potential.
There were four features that are especially important. Those features were already presented in
Chapter 2 to explain the generation of action potentials, but the reasons they were proposed by
Hodgkin and Huxley are explained below.
- The idea that ion channels have gates came from the observation that when the membrane
is depolarized, Na+ conductance first rises sharply, reaches a maximum and then falls back to its
resting value, even though the membrane potential did not change and was held constant at - 10
mV. Thus, Na
channels "open up" during the rising phase of the action potential and then
somehow automatically close again. The explanation they gave is that the Na
and K
channels
have gates associated with them.
- The Na
channel is viewed as having two gates; one that depolarization causes to swing
open, which they called the activation gate , and a second gate that is shut by depolarization,
which they called the inactivation gate. Since the positions of both gates are determined by
membrane depolarization, the two gates must have different kinetics to produce the Na+
conductance curve in Fig. 7. Specifically, they proposed that the activation gate opens quickly
with depolarization and the closing of the inactivation gate follows just a fraction of a
millisecond later. Hence, the Na+ channel is conductive for just a brief instant, less than 1.0 ms,
and then is rendered non-conductive by the closing of the inactivation gate.
- The K+ also has an activation gate opened by depolarization. However, once the
conductance is activated and reaches a maximum value, it remains at that value for as long as the
membrane remains depolarized. Consequently, K+ channels only have an activation gate and
unlike Na+ channels, they do not have an inactivation gate. The activation gates of K+ channels
remain open as long as the membrane remains depolarized.
- Finally, it can be seen from the conductance graph in Fig. 7 that depolarization first
causes the Na+ conductance to rise steeply followed less than a millisecond later by a more
slowly rising K+ conductance. The activation gates of Na
channels react more quickly than
either the Na+ inactivation gate or the K
activation gate. The delay in the opening of K+
channels allows the initial opening of Na+ to completely dominate the membrane for less than a
millisecond, thereby evoking a large influx of Na+ with a minimum of K+ efflux. That is what
accounts for the upstroke of the action potential.
Fig. 8. State of Na+ and K+ channels are the four main phases of the action potential. The
positions of the gates are shown in the left panels, the separated Na+ (red) and K+ (blue) currents from
voltage clamp records are shown in the center panels, and the corresponding state of the membrane
potential is represented in the right panels. The double-headed arrows in the center panel indicate the
Na+ and K+ currents that occur in each phase. For example, in phase 2, the Na channels are fully open
while K+ channels are still closed. The large Na+ current at that point is indicated in the center panel, as
is the very small K+ current. The upstroke of the action potential, which the Na+ currents generate, is
shown in the record of the membrane potential on the far right.
Conductance changes with membrane potential
In the previous sections, we showed how Hodgkin and Huxlex were able to calculate how
both Na+ and K+ conductances changed over time after being activated by depolarizing the
membrane to - 10 mV. Here we show how Na and K conductances change with membrane
potential. To show how conductance varies with membrane potential, they obtained a family of
curves representing the Na
and K
conductances at different membrane potentials, as shown in
Fig.. In these experiments they were not interested in how conductances changed over time,
they already solved that question as we showed in the previous section. Rather the question they
asked was, what is the largest Na+ and K+ conductance that is evoked as the membrane potential
was clamped at progressively more depolarized values? Thus, what they did is to calculate Na
Fig. 10. Graph showing how maximum K+ and Na+ conductance varies with membrane
potential. The data plotted graphically here were obtained from the data shown in Fig. 9 and the
values of the conductances were calculated from Ohm’s law.
