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Neuroscience: Exploring the Brain, Study notes of Neuroscience

INTRODUCTION. Now we come to the signal that conveys information over distances in the nervous system—the action potential. As we saw in Chapter 3, ...

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The Action Potential
INTRODUCTION
PROPERTIES OF THE ACTION POTENTIAL
THE UPS AND DOWNS OF AN ACTION POTENTIAL
Box 4.1 Brain Food: Methods of Recording Action Potentials
THE GENERATION OF AN ACTION POTENTIAL
THE GENERATION OF MULTIPLE ACTION POTENTIALS
THE ACTION POTENTIAL, IN THEORY
MEMBRANE CURRENTS AND CONDUCTANCES
THE INS AND OUTS OF AN ACTION POTENTIAL
THE ACTION POTENTIAL, IN REALITY
THE VOLTAGE-GATED SODIUM CHANNEL
Sodium Channel Structure
Functional Properties of the Sodium Channel
Box 4.2 Brain Food: The Patch-Clamp Method
The Effects of Toxins on the Sodium Channel
Box 4.3 Path of Discovery: Tet rod ot oxi n and th e Daw n of I on
Channel Pharmacology, by Toshio Narahashi
VOLTAGE-GATED POTASSIUM CHANNELS
PUTTING THE PIECES TOGETHER
ACTI ON POTENT IAL CONDUCT ION
FACTOR S I NFLUENC ING CONDUCTI ON VEL OCITY
Box 4.4 Of Special Interest: Local Anesthesia
MYELIN AND SALTATORY CONDUCTION
Box 4.5 Of Special Interest: Multiple Sclerosis, a
Demyelinating Disease
ACTI ON POTENT IALS, AXONS, AND DENDRITES
Box 4.6 Of Special Interest: The Eclectic Electric Behavior
of Neurons
CONCLUDING REMARKS
CHAPTER
4
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The Action Potential

INTRODUCTION

PROPERTIES OF THE ACTION POTENTIAL

THE UPS AND DOWNS OF AN ACTION POTENTIAL

■ Box 4.1 Brain Food: Methods of Recording Action Potentials THE GENERATION OF AN ACTION POTENTIAL THE GENERATION OF MULTIPLE ACTION POTENTIALS

THE ACTION POTENTIAL, IN THEORY

MEMBRANE CURRENTS AND CONDUCTANCES

THE INS AND OUTS OF AN ACTION POTENTIAL

THE ACTION POTENTIAL, IN REALITY

THE VOLTAGE-GATED SODIUM CHANNEL

Sodium Channel Structure Functional Properties of the Sodium Channel ■ Box 4.2 Brain Food: The Patch-Clamp Method The Effects of Toxins on the Sodium Channel ■ Box 4.3 Path of Discovery: Tetrodotoxin and the Dawn of Ion Channel Pharmacology, by Toshio Narahashi VOLTAGE-GATED POTASSIUM CHANNELS PUTTING THE PIECES TOGETHER

ACTION POTENTIAL CONDUCTION

FACTORS INFLUENCING CONDUCTION VELOCITY

■ Box 4.4 Of Special Interest: Local Anesthesia MYELIN AND SALTATORY CONDUCTION ■ Box 4.5 Of Special Interest: Multiple Sclerosis, a Demyelinating Disease

ACTION POTENTIALS, AXONS, AND DENDRITES

■ Box 4.6 Of Special Interest: The Eclectic Electric Behavior of Neurons

CONCLUDING REMARKS

C H A P T E R

▼ INTRODUCTION

Now we come to the signal that conveys information over distances in the nervous system—the action potential. As we saw in Chapter 3, the cytosol in the neuron at rest is negatively charged with respect to the extracellular fluid. The action potential is a rapid reversal of this situation such that, for an instant, the inside of the membrane becomes positively charged with respect to the outside. The action potential is also often called a spike, a nerve impulse, or a discharge. The action potentials generated by a cell are all similar in size and dura- tion, and they do not diminish as they are conducted down the axon. Keep in mind the big picture: The frequency and pattern of action potentials con- stitute the code used by neurons to transfer information from one location to another. In this chapter, we discuss the mechanisms that are responsible for the action potential and how it propagates down the axonal membrane.

▼ PROPERTIES OF THE ACTION POTENTIAL

Action potentials have certain universal properties, features that are shared by axons in the nervous systems of every beast, from a squid to a college student. Let’s begin by exploring some of these properties. What does the action potential look like? How is it initiated? How rapidly can a neuron generate action potentials?

The Ups and Downs of an Action Potential

In Chapter 3, we saw that the membrane potential, Vm, can be determined by inserting a microelectrode in the cell. A voltmeter is used to measure the electrical potential difference between the tip of this intracellular micro- electrode and another placed outside the cell. When the neuronal mem- brane is at rest, the voltmeter reads a steady potential difference of about –65 mV. During the action potential, however, the membrane potential briefly becomes positive. Because this occurs so rapidly—100 times faster than the blink of an eye—a special type of voltmeter, called an oscilloscope , is used to study action potentials. The oscilloscope records the voltage as it changes over time (Box 4.1). An action potential, as it would appear on the display of an oscilloscope, is shown in Figure 4.1. This graph represents a plot of membrane potential versus time. Notice that the action potential has certain identifiable parts. The first part, called the rising phase , is characterized by a rapid depolar- ization of the membrane. This change in membrane potential continues until V (^) m reaches a peak value of about 40 mV. The part of the action po- tential where the inside of the neuron is positively charged with respect to the outside is called the overshoot. The falling phase of the action potential is a rapid repolarization until the membrane is actually more neg- ative than the resting potential. The last part of the falling phase is called the undershoot , or after-hyperpolarization. Finally, there is a gradual restoration of the resting potential. From beginning to end, the action potential lasts about 2 milliseconds (msec).

The Generation of an Action Potential

In Chapter 3, we said that breaking of the skin by a thumbtack was sufficient to generate action potentials in a sensory nerve. Let’s use this example to see how an action potential begins. The perception of sharp pain when a thumbtack enters your foot is caused by the generation of action potentials in certain nerve fibers in the

76 C H A P T E R 4 • THE ACTION POTENTIAL

78 C H A P T E R 4 • THE ACTION POTENTIAL

Methods for studying nerve impulses may be broadly divided into two types: intracellular and extracellular (Figure A). Intracellular recording requires impaling the neuron or axon with a microelectrode. The small size of most neurons makes this method challenging, and this explains why so many of the early studies on action potentials were per- formed on the neurons of invertebrates, which can be 50–100 times larger than mammalian neurons. Fortu- nately, recent technical advances have made even the smallest vertebrate neurons accessible to intracellular recording methods, and these studies have confirmed that much of what was learned in invertebrates is directly ap- plicable to humans. The goal of intracellular recording is simple: to meas- ure the potential difference between the tip of the intra- cellular electrode and another electrode placed in the solution bathing the neuron (continuous with the earth, and thus called ground). The intracellular electrode is filled with a concentrated salt solution (often KCl) having a high electrical conductivity. The electrode is connected to an amplifier that compares the potential difference between this electrode and ground.This potential difference can be displayed using an oscilloscope. The oscilloscope sweeps a beam of electrons from left to right across a phosphor screen.Vertical deflections of this beam can be read as changes in voltage. The oscilloscope is really just a sophisticated voltmeter that can record rapid changes in voltage (such as an action potential). As we shall see in this chapter, the action potential is characterized by a sequence of ionic movements across the neuronal membrane. These electri- cal currents can be detected without impaling the neuron by placing an elec- trode near the membrane. This is the principle behind extracellular recording. Again, we measure the potential differ-

ence between the tip of the recording electrode and ground. The electrode can be a fine glass capillary filled with a salt solution, but it is often simply a thin insulated metal wire. Normally, in the absence of neural activity, the potential difference between the extracellular recording electrode and ground is zero. However, when the action potential arrives at the recording position, positive charges flow away from the recording electrode, into the neuron. Then, as the action potential passes by, positive charges flow out across the membrane toward the recording elec- trode. Thus, the extracellular action potential is charac- terized by a brief, alternating voltage difference between the recording electrode and ground. (Notice the different scale of the voltage changes produced by the action potential recorded with intracellular and extracellular recordings.) These changes in voltage can be seen using an oscilloscope, but they can also be heard by connecting the output of the amplifier to a loudspeaker. Each impulse makes a distinctive “pop” sound. Indeed, recording the activity of an active sensory nerve sounds just like mak- ing popcorn.

Box 4.1^ B R A I N^ F O O D

Methods of Recording Action Potentials

Amplifier

Oscilloscope display

Ground Intracellular electrode

Extracellular electrode

–60 μV

–40 μV

–20 μV

0 μV

20 μV

40 μV

–60 mV

–40 mV

–20 mV

0 mV

20 mV

40 mV

FIGURE A

that fashion and sports photographers use? In that case, continued pressure on the shutter button beyond threshold would cause the cam- era to shoot frame after frame. The same thing is true for a neuron. If, for example, we pass continuous depolarizing current into a neuron through a microelectrode, we will generate not one, but many action potentials in succession (Figure 4.2).

The rate of action potential generation depends on the magnitude of the continuous depolarizing current. If we pass enough current through a microelectrode to depolarize just to threshold, but not far beyond, we might find that the cell generates action potentials at a rate of something like one per second, or 1 hertz (Hz). If we crank up the current a little bit more, however, we will find that the rate of action potential generation increases, say, to 50 impulses per second (50 Hz). Thus, the firing frequency of action potentials reflects the magnitude of the depolarizing current. This is one way that stimulation intensity is encoded in the nervous system (Figure 4.3). Although firing frequency increases with the amount of depolarizing current, there is a limit to the rate at which a neuron can generate action potentials. The maximum firing frequency is about 1000 Hz; once an action

FIGURE 4. The effect of injecting positive charge into a neuron. (a) The axon hillock is impaled by two electrodes, one for recording the membrane potential relative to ground and the other for stimulating the neuron with electrical current. (b) When electrical current is injected into the neuron (top trace), the membrane is depolarized sufficiently to fire action potentials (bottom trace).

▼ PROPERTIES OF THE ACTION POTENTIAL 79

0

0

Injected Injected current current

Amplifier

Ground

Recording Stimulating electrode electrode

Axon

40

  • Time

Membrane potential (mV)–

(a) (b)

–65 mV

0

0

Time

Injectedcurrent

If injected current does not depolarize the membrane to threshold, no action potentials will be generated.

If injected current depolarizes the mem- brane beyond threshold, action potentials will be generated.

The action potential firing rate increases as the depolarizing current increases.

FIGURE 4. The dependence of action potential firing frequency on the level of depolarization.

Now let’s take another look at our example. Initially, we began with V (^) m! 0 mV and no ionic membrane permeability (Figure 4.4a). There is a large driving force on K"^ ions because Vm # EK; in fact, (Vm $ EK)! 80 mV. However, because the membrane is impermeable to K", the potassium con- ductance, g (^) K , equals zero. Consequently, I (^) K! 0. Potassium current only flows when we stipulate that the membrane has open potassium channels, and therefore g (^) K % 0. Now K "^ ions flow out of the cell—as long as the

FIGURE 4. Membrane currents and conductances. Here is an ideal neuron with sodium-potassium pumps (not shown), potassium channels, and sodium channels. The pumps establish ionic concentration gradients so that K"^ is concentrated inside the cell and Na"^ is concentrated outside the cell. (a) Initially, we assume that all channels are closed and the membrane potential equals 0 mV. (b) Now we open the potassium channels, and K "^ flows out of the cell. This movement of K "^ is an electrical current, IK, and it flows as long as the membrane conductance to K "^ ions, gK, is greater than zero, and the membrane potential is not equal to the potassium equilibrium potential. (c) At equilibrium, there is no net potassium current because, although g (^) K % 0, the membrane potential at equilibrium equals EK. At equilibrium, an equal number of K "^ ions enters and leaves.

▼ THE ACTION POTENTIAL, IN THEORY 81

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

KK++^ KK++

KK++

K+^ K +

K +^ K +

K+^ K +

K +^ K +

+

-

+

-

+

-

+

-

Outside cell

Outside

Inside cell

Inside

Ideal neuron

Sodium channel

Potassium channel

Vm 0

EK = – 80 mV E (^) Na = 62 mV gK = 0 I (^) K = g (^) K (Vm– EK) = 0

(a)

(b)

(c)

V (^) m 0

EK = – 80 mV E (^) Na = 62 mV gK > 0 I (^) K = g (^) K (Vm– EK) > 0

V (^) m 0

  • 80

EK = – 80 mV E (^) Na = 62 mV gK > 0 I (^) K = g (^) K (Vm– EK) = 0

membrane potential differs from the potassium equilibrium potential (Figure 4.4b). Notice that the current flow is in the direction that takes Vm toward EK. When Vm! EK, the membrane is at equilibrium, and no net current will flow. In this condition, although there is a large potassium conductance, gK, there is no longer any net driving force on the K"^ ions (Figure 4.4c).

The Ins and Outs of an Action Potential

Let’s pick up the action where we left off in the last section. The membrane of our ideal neuron is permeable only to K "^ , and V (^) m! E (^) K! #80 mV. What’s happening with the Na"^ ions concentrated outside the cell? Because the membrane potential is so negative with respect to the sodium equilib- rium potential, there is a very large driving force on Na "^ ([V (^) m # E (^) Na ]! [#80 mV # 62 mV]! #142 mV). Nonetheless, there can be no net Na " current as long as the membrane is impermeable to Na". But now let’s open the sodium channels and see what happens to the membrane potential. At the instant we change the ionic permeability of the membrane, gNa is high, and, as we said above, there is a large driving force pushing on Na ". Thus, we have what it takes to generate a large sodium current, I (^) Na , across the membrane. Na "^ ions pass through the membrane sodium channels in the direction that takes Vm toward ENa ; in this case, the sodium current, INa , is inward across the membrane. Assuming the membrane permeability is now far greater to sodium than it is to potassium, this influx of Na " depolarizes the neuron until V (^) m approaches E (^) Na , 62 mV. Notice that something remarkable happened here. Simply by switching the dominant membrane permeability from K "^ to Na ", we were able to rapidly reverse the membrane potential. In theory, then, the rising phase of the action potential could be explained if, in response to depolarization of the membrane beyond threshold, membrane sodium channels opened. This would allow Na"^ to enter the neuron, causing a massive depolariza- tion until the membrane potential approached E (^) Na. How could we account for the falling phase of the action potential? Sim- ply assume that sodium channels quickly close and the potassium channels remain open, so the dominant membrane ion permeability switches back from Na "^ to K ". Then K"^ would flow out of the cell until the membrane potential again equals E (^) K. Notice that if g (^) K increased during the falling phase, the action potential would be even briefer. Our model for the ins and outs, ups and downs of the action potential in an ideal neuron is shown in Figure 4.5. The rising phase of the action potential is explained by an inward sodium current, and the falling phase is explained by an outward potassium current. The action potential there- fore could be accounted for simply by the movement of ions through channels that are gated by changes in the membrane potential. If you un- derstand this concept, you understand a lot about the ionic basis of the action potential. What’s left now is to see how this actually happens—in a real neuron.

▼ THE ACTION POTENTIAL, IN REALITY

Let’s quickly review our theory of the action potential. When the mem- brane is depolarized to threshold, there is a transient increase in gNa. The increase in g (^) Na allows the entry of Na"^ ions, which depolarizes the neuron. And the increase in g (^) Na must be brief in duration to account for the short duration of the action potential. Restoring the negative membrane poten- tial would be further aided by a transient increase in g (^) K during the falling phase, allowing K"^ ions to leave the depolarized neuron faster.

82 C H A P T E R 4 • THE ACTION POTENTIAL

Testing this theory is simple enough in principle. All one has to do is measure the sodium and potassium conductances of the membrane dur- ing the action potential. In practice, however, such a measurement proved to be quite difficult in real neurons. The key technical breakthrough was the introduction of a device called a voltage clamp , invented by the American physiologist Kenneth C. Cole, and the decisive experiments us- ing it were performed by Cambridge University physiologists Alan Hodgkin and Andrew Huxley around 1950. The voltage clamp enabled Hodgkin and Huxley to “clamp” the membrane potential of an axon at any value they chose. They could then deduce the changes in membrane conductance that occur at different membrane potentials by measuring the currents that flowed across the membrane. In an elegant series of experiments, Hodgkin and Huxley showed that the rising phase of the action potential was in- deed caused by a transient increase in g (^) Na and an influx of Na!^ ions, and that the falling phase was associated with an increase in g (^) K and an efflux of K!^ ions. Their accomplishments were recognized with the Nobel Prize in 1963. To account for the transient changes in g (^) Na , Hodgkin and Huxley pro- posed the existence of sodium “gates” in the axonal membrane. They hy- pothesized that these gates are “activated”—opened—by depolarization above threshold and “inactivated”—closed and locked—when the mem- brane acquires a positive membrane potential. These gates are “deinacti- vated”—unlocked and enabled to be opened again—only after the mem- brane potential returns to a negative value. It is a tribute to Hodgkin and Huxley that their hypotheses about mem- brane gates predated by more than 20 years the direct demonstration of voltage-gated channel proteins in the neuronal membrane. We have a new understanding of gated membrane channels, thanks to two more recent sci- entific breakthroughs. First, new molecular biological techniques have en- abled neuroscientists to determine the detailed structure of these proteins. Second, new neurophysiological techniques have enabled neuroscientists to measure the ionic currents that pass through single channels. We will now explore the action potential from the perspective of these membrane ion channels.

The Voltage-Gated Sodium Channel

The voltage-gated sodium channel is aptly named. The protein forms a pore in the membrane that is highly selective to Na!^ ions, and the pore is opened and closed by changes in the electrical potential of the membrane.

Sodium Channel Structure. The voltage-gated sodium channel is created from a single long polypeptide. The molecule has four distinct domains, numbered I–IV; each domain consists of six transmembrane alpha helices, numbered S1–S6 (Figure 4.6). The four domains are believed to clump to- gether to form a pore between them. The pore is closed at the negative rest- ing membrane potential. When the membrane is depolarized to threshold, however, the molecule twists into a configuration that allows the passage of Na!^ through the pore (Figure 4.7). Like the potassium channel, the sodium channel has pore loops that are assembled into a selectivity filter. This filter makes the sodium channel 12 times more permeable to Na!^ than it is to K !. Apparently, the Na!^ ions are stripped of most, but not all, of their associated water molecules as they pass into the channel. The retained water serves as a sort of molecular chap- erone for the ion, and is necessary for the ion to pass the selectivity filter.

84 C H A P T E R 4 • THE ACTION POTENTIAL

FIGURE 4. The structure of the voltage-gated sodium channel. (a) A depiction of how the sodium channel polypeptide chain is believed to be woven into the membrane. The mole- cule consists of four domains, I–IV. Each domain consists of six alpha helices (represented by the blue cylinders), which pass back and forth across the membrane. (b) An expanded view of one domain, showing the voltage sensor of alpha helix S4 and the pore loop (red), which contributes to the selectivity filter. (c) A view of the molecule showing how the domains may arrange themselves to form a pore between them. (Source: Adapted from Armstrong and Hille, 1998, Fig. 1.)

▼ THE ACTION POTENTIAL, IN REALITY 85

++ ++

++ ++

++ ++

++ ++

(a)

(b)

(c)

S1 S2 S S4 S5 S

Inside cell

N

C

Outside cell I^ II^ III^ IV

Pore loop

Gate

Voltage sensor

Selectivity filter

to see how many of the properties of the action potential can be ex- plained by the properties of the voltage-gated sodium channel. For exam- ple, the fact that single channels do not open until a critical level of membrane depolarization is reached explains the action potential thresh- old. The rapid opening of the channels in response to depolarization explains why the rising phase of the action potential occurs so quickly. And the

FIGURE 4. The opening and closing of sodium channels upon membrane depolarization. (a) This trace shows the electrical potential across a patch of membrane. When the mem- brane potential is changed from !65 to !40 mV, the sodium channels pop open. (b) These traces show how three different channels respond to the voltage step. Each line is a record of the electrical current that flows through a single channel. ➀ At !65 mV, the channels are closed, so there is no current. ➁ When the membrane is depolarized to !40 mV, the chan- nels briefly open and current flows inward, represented by the downward deflection in the current traces. Although there is some variability from channel to channel, all of them open with little delay and stay open for less than 1 msec. Notice that after they have opened once, they close and stay closed as long as the membrane is maintained at a depolarized Vm. ➂ The closure of the sodium channel by steady depolarization is called inactivation. ➃ To deinactivate the channels, the membrane must be returned to !65 mV again. (c) A model for how changes in the conformation of the sodium channel protein might yield its functional properties. ➀ The closed channel ➁ opens upon membrane depolarization. ➂ Inactivation occurs when a globular portion of the protein swings up and occludes the pore. ➃ Deinacti- vation occurs when the globular portion swings away and the pore closes by movement of the transmembrane domains.

▼ THE ACTION POTENTIAL, IN REALITY 87

(a)

Na +

1 2

3

(b)

(c)

Channel closed

Channel open

Inward current

V (^) m

  • 40 mV

5 msec

  • 65 mV

4 Inward current

Inward current

Sodium channel

1 2 3 4

Membrane

88 C H A P T E R 4 • THE ACTION POTENTIAL

The very existence of voltage-gated channels in the neu- ronal membrane was merely conjecture until the develop- ment of methods to study individual channel proteins. A revolutionary new method, the patch clamp, was developed by German neuroscientists Bert Sakmann and Erwin Neher in the mid-1970s. In recognition of their contribution, Sakmann and Neher were awarded the 1991 Nobel Prize. Patch clamping enables one to record ionic currents through single channels (Figure A). The first step is gently lowering the fire-polished tip of a glass recording elec- trode, 1–5 μm in diameter, onto the membrane of the neuron (part a), and then applying suction through the electrode tip (part b). A tight seal forms between the walls of the electrode and the underlying patch of mem- brane. This “gigaohm” seal (so named because of its high electrical resistance,! 109 Ω) leaves the ions in the elec-

trode only one path to take, through the channels in the underlying patch of membrane. If the electrode is then withdrawn from the cell, the membrane patch can be torn away (part c), and ionic currents can be measured as steady voltages are applied across the membrane (part d). With a little luck, one can resolve currents flowing through single channels. If the patch contains a voltage- gated sodium channel, for example, then changing the membrane potential from "65 to "40 mV will cause the channel to open, and current (I) will flow through it (part e). The amplitude of the measured current at a constant membrane voltage reflects the channel conductance, and the duration of the current reflects the time the channel is open. Patch-clamp recordings reveal that most channels flip between two conductance states that can be interpreted as open or closed.The time they remain open can vary, but the single-channel conductance value stays the same and is there- fore said to be unitary. Ions can pass through single channels at an astonishing rate—well over a million per second.

Box 4.2^ B R A I N^ F O O D

The Patch-Clamp Method

(b) (c) (d)

(a)

(e)

Pipette tip

Sodium channel (closed)

Voltage change across a patch of membrane

Sodium channel (open)

Neuron

Pipette

Vm

Channel open Channel closed

Out

I

In

–65 mV

Gigaohm seal

Na+

FIGURE A

90 C H A P T E R 4 • THE ACTION POTENTIAL

Puffer fish is regarded as the most delicious fish in Japan (Figure A). However, the tetrodotoxin (TTX) it contains makes the fish very dangerous to eat, and a special license is required to serve puffer fish at a restaurant in Japan.Yet some “fish lovers” try to achieve ecstasy through the numb feeling on the lips that comes from eating a small piece of ovary or liver containing TTX. This sometimes results in accidental death, which is caused by paralysis of the diaphragm due to nerve and muscle block. TTX has now been a source of scientific ecstasy in neurophysiology. Shortly after starting my scientific career at the Uni- versity of Tokyo, I came across fascinating papers by Hodgkin, Huxley, and Katz in which they extensively uti- lized the voltage clamp technique originally invented by Cole. This was the dawn of the ion channel theory of nerve excitation. Since that time, I have cherished a dream of explaining the mechanism of action of various drugs in terms of changes in ion channel function. However, voltage clamp was an extremely difficult technique at that time.

In 1959, I encountered a very specific and potentially important action of the puffer fish toxin TTX. Using the intracellular microelectrode recording of action potentials from frog skeletal muscle, we found that TTX blocked action potentials through selective inhibition of sodium channels without any changes in potassium channels. However, the ultimate conclusion awaited voltage clamp experimentation. I reported the TTX study at the Japanese Pharmacology Society Meeting in Tokyo in 1960. There were only two young pharmacologists in the audience who were familiar with ion channels, and intense discussions ensued. Drs. Masanori Otsuka and Makoto Endo have remained very good friends since that time. The day I left for the United States in 1961, Dr. Norimoto Urakawa, a collaborator in the TTX study, slipped a small vial of TTX in my pocket. We were hoping that someday we would be able to prove the validity of our hypothesis of TTX action by the voltage clamp technique. The chance finally arrived in late 1962 when I was at Duke University Medical Center. Dr. John W. Moore, an expert in voltage clamp, and I thought we could finish the TTX experiments before my return to Japan to obtain an immigrant visa. Experiments using lobster giant axons were performed literally day and night during the Christmas season, with the help of William Scott (then a medical student). The technique was extremely difficult, yet we managed to obtain results sufficient for publication. I took the freshly developed 35 mm films of ionic current records, which had barely dried (no computer at that time), to Japan for analysis. After submitting a manuscript, I re- ceived the first request for a TTX sample—jotted down with the signature at the end of the referee’s comments. This 1964 paper, clearly demonstrating TTX’s selective and potent block of sodium channels, marked the begin- ning of a new era. In the early 1960s, it was inconceivable to utilize any chemicals and toxins as tools for the study of ion channel function. TTX has since been used as a popular chemical tool to characterize sodium channels and other channels, because of its highly specific action. The TTX study indeed opened a new concept of studying the mechanism of action of various drugs, toxins, and chem- icals on neuronal receptors and ion channels, a neuro- science field now flourishing in biomedical science.

Box 4.3^ P A T H^ O F^ D I S C O V E R Y

Tetrodotoxin and the Dawn

of Ion Channel Pharmacology

by Toshio Narahashi

FIGURE A A puffer fish blows out when irritated. (Courtesy of Dr.T. Narahashi.)

Voltage-Gated Potassium Channels

Hodgkin and Huxley’s experiments indicated that the falling phase of the action potential was explained only partly by the inactivation of gNa. They found there was also a transient increase in g (^) K that functioned to speed the restoration of a negative membrane potential after the spike. They pro- posed the existence of membrane potassium gates that, like sodium gates, open in response to depolarization of the membrane. Unlike sodium gates, however, potassium gates do not open immediately upon depolarization; it takes about 1 msec for them to open. Because of this delay, and because this potassium conductance serves to rectify, or reset, the membrane potential, they called this conductance the delayed rectifier. We now know that there are many different types of voltage-gated potas- sium channels. Most of them open when the membrane is depolarized and function to diminish any further depolarization by giving K!^ ions a path to leave the cell across the membrane. The known voltage-gated potassium channels have a similar structure. The channel proteins consist of four separate polypeptide subunits that come together to form a pore be- tween them. Like the sodium channel, these proteins are sensitive to changes in the electrical field across the membrane. When the membrane is depolarized, the subunits are believed to twist into a shape that allows K!^ ions to pass through the pore.

Putting the Pieces Together

We can now use what we’ve learned about ions and channels to explain the key properties of the action potential (Figure 4.10).

Threshold. Threshold is the membrane potential at which enough volt- age-gated sodium channels open so that the relative ionic permeability of the membrane favors sodium over potassium.

Rising phase. When the inside of the membrane has a negative electrical potential, there is a large driving force on Na!^ ions. Therefore, Na!^ ions rush into the cell through the open sodium channels, causing the mem- brane to rapidly depolarize.

Overshoot. Because the relative permeability of the membrane greatly favors sodium, the membrane potential goes to a value close to E (^) Na , which is greater than 0 mV.

Falling phase. The behavior of two types of channel contributes to the falling phase. First, the voltage-gated sodium channels inactivate. Sec- ond, the voltage-gated potassium channels finally open (triggered to do so 1 msec earlier by the depolarization of the membrane). There is a great driving force on K!^ ions when the membrane is strongly depolar- ized. Therefore, K!^ ions rush out of the cell through the open channels, causing the membrane potential to become negative again.

Undershoot. The open voltage-gated potassium channels add to the rest- ing potassium membrane permeability. Because there is very little sodium permeability, the membrane potential goes toward E (^) K, causing a hyper- polarization relative to the resting membrane potential until the voltage- gated potassium channels close again.

Absolute refractory period. Sodium channels inactivate when the mem- brane becomes strongly depolarized. They cannot be activated again, and another action potential cannot be generated, until the membrane potential goes sufficiently negative to deinactivate the channels.

▼ THE ACTION POTENTIAL, IN REALITY 91

remember that the sodium-potassium pump also is working quietly in the background. Imagine that the entry of Na!^ during each action po- tential is like a wave coming over the bow of a boat making way in heavy seas. Like the continuous action of the boat’s bilge pump, the sodium-potassium pump works all the time to transport Na!^ back across the membrane. The pump maintains the ionic concentration gradients that drive Na!^ and K!^ through their channels during the action potential.

▼ ACTION POTENTIAL CONDUCTION

In order to transfer information from one point to another in the nervous system, it is necessary that the action potential, once generated, be conducted down the axon. This process is like the burning of a fuse. Imagine you’re holding a firecracker with a burning match held under the fuse. The fuse ignites when it gets hot enough (beyond some threshold). The tip of the burning fuse heats up the segment of fuse immediately ahead of it until it ignites. In this way, the flame steadily works its way down the fuse. Note that the fuse lit at one end only burns in one direction; the flame cannot turn back on itself because the combustible material just behind it is spent. Propagation of the action potential along the axon is similar to the prop- agation of the flame along the fuse. When the axon is depolarized suffi- ciently to reach threshold, voltage-gated sodium channels open, and the action potential is initiated. The influx of positive charge depolarizes the segment of membrane immediately before it until it reaches threshold and generates its own action potential (Figure 4.11). In this way, the action potential works its way down the axon until it reaches the axon terminal, thereby initiating synaptic transmission (the subject of Chapter 5). An action potential initiated at one end of an axon propagates only in one direction; it does not turn back on itself. This is because the membrane just behind it is refractory, due to inactivation of the sodium channels. But, just like the fuse, an action potential can be generated by depolarization at either end of the axon and therefore can propagate in either direction. (Normally, action potentials conduct only in one direction; this is called orthodromic conduction. Backward propagation, sometimes elicited experimentally, is

▼ ACTION POTENTIAL CONDUCTION 93

+^ +

+

+

+

Time zero

1 msec later

2 msec later

3 msec later

FIGURE 4. Action potential conduction. The entry of positive charge during the action potential causes the membrane just ahead to depolarize to threshold.

called antidromic conduction.) Note that because the axonal membrane is excitable (capable of generating action potentials) along its entire length, the impulse will propagate without decrement. The fuse works the same way, because it is combustible along its entire length. Unlike the fuse, how- ever, the axon can regenerate its firing ability. Action potential conduction velocities vary, but 10 m/sec is a typical rate. Remember, from start to finish the action potential lasts about 2 msec. From this, we can calculate the length of membrane that is engaged in the action potential at any instant in time: 10 m/sec! 2! 10 "^3 sec # 2! 10 "^2 m. Therefore, an action potential traveling at 10 m/sec occurs over a 2 cm length of axon.

Factors Influencing Conduction Velocity

Remember that the inward Na$^ current during the action potential depolar- izes the patch of membrane just ahead. If this patch reaches threshold, it will fire an action potential, and the action potential will “burn” on down the membrane. The speed with which the action potential propagates down the axon depends on how far the depolarization ahead of the action potential spreads, which in turn depends on certain physical characteristics of the axon. Imagine that the influx of positive charge into an axon during the action potential is like turning on the water to a leaky garden hose. There are two paths the water can take: one, down the inside of the hose; the other, across the hose through the leaks. How much water goes along each path depends on their relative resistance; most of the water will take the path of least resistance. If the hose is narrow and the leaks are numerous and large, most of the water will flow out through the leaks. If the hose is wide and the leaks are few and tiny, most of the water will flow down the in- side of the hose. The same principles apply to positive current spreading down the axon ahead of the action potential. There are two paths that positive charge can take: one, down the inside of the axon; the other, across the axonal membrane. If the axon is narrow and there are many open membrane pores, most of the current will flow out across the membrane. If the axon is wide and there are few open membrane pores, most of the current will flow down inside the axon. The farther the current goes down the axon, the farther ahead of the action potential the membrane will be depolarized, and the faster the action poten- tial will propagate. As a rule, therefore, action potential conduction velocity increases with increasing axonal diameter. As a consequence of this relationship between axonal diameter and conduction velocity, neural pathways that are especially important for sur- vival have evolved unusually large axons. An example is the giant axon of the squid, which is part of a pathway that mediates an escape reflex in response to strong sensory stimulation. The squid giant axon can be 1 mm in diameter, so large that originally it was thought to be part of the ani- mal’s circulatory system. Neuroscience owes a debt to British zoologist J. Z. Young, who, in 1939, called attention to the squid giant axon as an exper- imental preparation for studying the biophysics of the neuronal membrane. Hodgkin and Huxley used this preparation to elucidate the ionic basis of the action potential, and the giant axon continues to be used today for a wide range of neurobiological studies. It is interesting to note that axonal size, and the number of voltage-gated channels in the membrane, also affect axonal excitability. Smaller axons require greater depolarization to reach action potential threshold and are more sensitive to being blocked by local anesthetics (Box 4.4).

94 C H A P T E R 4 • THE ACTION POTENTIAL