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9

Propagated Signaling: The Action Potential

The Action Potential Is Generated by the Flow of Mutations in -GatedChannels Cause Specific Through Voltage-Gated Channels Neurological Diseases and PotassiumCurrents Through Voltage-Gated An Overall View ChannelsAre RecordedWith the VoltageClamp Voltage-GatedSodium and PotassiumConductances Are Calculated From Their Currents ERVECELLS ARE ABLE TO carry signalsover long The Action Potential Can Be ReconstructedFrom the N Propertiesof Sodium and PotassiumChannels distancesbecause of their ability to generatean Variations in the Properties of Voltage-Gated Channels action potential-a regenerative electrical sig- Increasethe Signaling Capabilities of nal whose amplitude does not attenuate as it moves down the . In Chapter 7 we saw how an action p0- The Nervous SystemExpresses a Rich Variety of Voltage- tential arises from sequential changes in the mem- Gated Ion Channels brane'sselective penneability to Na+ and K+ ions. In Gating of Voltage-SensitiveIon ChannelsCan Be Chapter 8 we considered how the membrane's passive Influenced by Various Cytoplasmic Factors properties influence the speed at which action poten- Excitability PropertiesVary BetweenRegions of the tials are conducted. In this chapter we focus on the voltage-gated ion channels that are critical for generat- Excitability PropertiesVary Among Neurons ing and propagating action potentials and consider how The Signaling Functions of Voltage-Gated Channels Can Be thesechannels are responsiblefor many important fea- Related to Their Molecular Structures tures of a neuron's electrical excitability. Opening of Voltage-GatedChannels Is All-or-None Redistribution of ChargesWithin Voltage-GatedSodium ChannelsControls Channel Gating The Action Potential Is Generated by the Flow of Ions Through Voltage-Gated Channels The Voltage-GatedSodium Channel Selectsfor Sodium on the Basisof Size,Charge, and Energy of Hydration of the Ion An important early clue about how action potentials are Genes Encoding the , Sodium, and generated came from an experiment performed by Ken- Channels Stem From a Common Ancestor neth Cole and Howard Curtis. While recording from the giant axon of the they found that the ion conduc- Various Smaller Subunits Contribute to the Functional Properties of Voltage-GatedSodium, Calcium, and tance across the membrane increasesdramatically dur- PotassiumChannels ing the action potential (Figure 9-1). This discovery pro- The Diversity of Voltage-GatedChannel Types Is Due to vided the first evidence that the action potential results SeveralGenetic Mechanisms from changesin the flux of ions through the channels of

~ Chapter 9 / PropagatedSignaling: The Action Potential 151

the membrane. It also raised the question: Which ions are responsible for the action potential? A key to this problem was provided by Alan Ionic conductance Hodgkin and , who found that the ampli- ./ tude of the action potential is reduced when the external Na+ concentrationis lowered, indicating that Na+ in- flux is responsible for the rising phase of the action po- tential. Their data also suggested that the falling phase of the action potential was caused by a later increase in K+ permeability. Hodgkin and Katz proposed that de- polarization of the above threshold causesa brief in- creasein the 's permeability to Na +, dur- Figure 9.1 A net increasein ionic conductancein the mem- ing which Na + permeability overwhelms the dominant brane of the axon accompanies the action potential. This historic recording from an experiment conducted in 1938 by permeability of the resting cell membrane to K+ ions. Kenneth Cole and Howard Curtis shows the oscilloscope record of an action potential superimposed on a simultaneous record of the ionic conductance. Sodium and Potassium Currents Through Voltage-Gated Channels Are Recorded With the To test this hypothesis, Hodgkin and Andrew Hux- nels. The voltage clamp does so by injecting a current ley conducted a second series of experiments. They sys- into the axon that is equal and opposite to the current tematically varied the in the squid flowing through the voltage-gated membrane channels. giant axon and measured the resulting changes in the In this way the voltage clamp prevents the charge sepa- membraneconductance to Na+ and K+ through volt- ration acrossthe membrane from changing. The amount age-gated Na + and K+ channels. To do this they made of current that must be generatedby the voltage clamp to use of a new apparatus, the voltage clamp. Prior to the keep the membrane potential constant provides a direct availability of the voltage-damp technique, attempts to measure of the current flowing across the membrane measureNa + and K+ conductanceas a function of (Box 9-1). Using the voltage-clamp technique, Hodgkin membrane potential had been limited by the strong and Huxley provided the first complete description of interdependence of the membrane potential and the the ionic mechanismsunderlying the action potential. gating of Na+ and K+ channels.For example,if the One advantage of the voltage clamp is that it readily membrane is depolarized sufficiently to open some of allows the total membrane current to be separated into the voltage-gatedNa + channels,inward Na+ current its ionic and capacitive components. As described in flows through these channels and causesfurther depo- Chapter 8, the membrane potential V m, is proportional larization. The additional causes still to the charge Qm on the membrane (Cm). more Na+ channelsto open and consequentlyinduces When V m is not changing, Qm is constant and no capac- more inward Na + current: itive current (~Qm/ ~t) flows. Capacitive current flows only when V m is changing.Therefore, when the mem- ~ Depolarization ~ brane potential changesin response to a very rapid step of command potential, capacitive current flows only at Inward ~. OpenNa+ the beginning and end of the step. Since the capacitive current is essentially instantaneous, the ionic currents that flow through the gated membrane channels can be ~nelS analyzed separately. This cycle, which eventually drives Measurements of these ionic membrane currents the membrane potential to the peak of the action poten- can be used to calculate the voltage and time depen- tial, makes it impossible to achieve a stable membrane dence of changesin membrane conductancescaused by potential. A similar coupling between current and mem- the opening and closing of Na+ and K+ channels. This brane potential complicates the study of the voltage- information provides insights into the properties of gated K+ channels. these two types of channels. The basic function of the voltage clamp is to inter- A typical voltage-clamp experiment starts with the rupt the interaction between the membrane potential membrane potential clamped at its resting value. H a and the opening and closing of voltage-gated ion chan- 10 mV depolarizing potential step is commanded, we

Chapter 9 / Propagated Signaling: The Action Potential 153

observe that an initial, very brief outward current in- stantaneously discharges the membrane capacitanceby the amount required for a 10 mV depolarization. This capacitivecurrent (Ie) is followed by a smaller outward Outward ionic current that persists for the duration of this pulse. 1m 0 l~~ ~~ t- At the end of the pulse there is a brief inward capacitive Inward I 1/ current, and the total membrane current returns to zero (Figure 9-3A). The steady ionic current that persists B throughout the depolarization is the current that flows 0 through the resting ion channels of the membrane (see Vm Chapter 6) and is called the leakagecurrent, II' The total conductanceof this population of channels is called the -60 leakageconductance (gl)' These resting channels, which are always open, are responsible for generating the rest- ing membrane potential (see Chapter 7). In a typical neuron most of the resting channels are permeable to K+ ions; the remaining channels are permeable to 0- or Na+ ions. 1m If a larger depolarizing step is commanded, the cur- rent record becomes more complicated. The capacitive and leakage currents both increase in amplitude. In ad- dition, shortly after the end of the capacitive current and the start of the leakagecurrent, an inward current devel- ops; it reaches a peak within a few milliseconds, de- C clines, and gives way to an outward current. This out- ward current reaches a plateau that is maintained for the duration of the pulse (Figure 9-3B). Vm A simple interpretation of these results is that the depolarizing voltage step sequentially turns on active conductancechannels for two separate ions: one type of channel for inward current and another for outward current. Becausethese two oppositely directed currents partially overlap in time, the most difficult task in ana- lyzing voltage-clamp experiments is to determine their 0 separatetime courses. 1m Hodgkin and Huxley achieved this separation by changing ions in the bathing solution. By substituting a larger, impermeant cation (choline.H+) for Na +, they eliminated the inward Na + current. Subsequently, the 1 ms Time-- task of separating inward and outward currents was made easier by selectively blocking the voltage- Figure 9-3 A voltage-clampexperiment demonstrates the sensitive conductance channels with drugs or toxins. sequential activation of two types of voltage-gated chan- , a poison from certain Pacific puffer fish, nels. blocks the voltage-gated Na + channel with a very high A. A small depolarizationis accompaniedby capacitiveand potency in the nanomolar range of concentration. leakagecurrents (Ieand 'I, respectively). (Ingestion of only a few milligrams of tetrodotoxin from B. A larger depolarizationresults in larger capacitiveand leak- improperly prepared puffer fish, consumed as the age currents, plus an inward current followed by an outward Japanesesushi delicacy , can be fatal.) The cation current. tetraethylammonium specifically blocks the voltage- C. Depolarizingthe cell in the presence of tetrodotoxin (which blocks the Na+ current) and again in the presenceof gated K+ channel (Figure 9-4). tetraethylammonium (which blocks the K+ current!. reveals When tetraethylammonium is applied to the axon the pure K+ and Na+ currents UKand 'N.. respectively)after to block the K+ channels, the total membrane current subtracting 'e and II- (Im) consists of Ie- II, and INa. The leakage conductance, 154 Part IT/ Cell andMolecular of the Neuron

Figure9-4 Drugsthat blockvoltage-gated Na + and K+ channels. Tetrodotoxinand saxitoxin both bind to Na+ channelswith a very high affinity. Tetrodotoxin is produced by certain puffer fish, newts, and . Saxitoxin is synthesized by the di- noflagellatesGonyaulax that are responsible for red tides. Consumption of clams or other shellfish that have fed on the dinoflagellatesduring a red tide causes paralytic shellfish poisoning. , the ac- tive substance isolated from coca leaves, was the first substanceto be used as a local . It also blocks Na+ channels but with a lower affinity and specificity than tetrodotoxin. Tetraethylammo- Tetraethylammonium nium is a cation that blocks certain voltage-gatedK+ channelswith a relatively low affinity. The red plus signs represent positive charge.

gl1 is constant; it does not vary with V m or with time. ization. At all levels of depolarization the Na + channe] Therefore, 111the leakage current, can be readily calcu- open more rapidly than do the K+ channels(Figure 9-6 lated and subtracted from 1m/leaving INa and Ic. Because When the depolarization is maintained for some timl Ic occurs only briefly at the beginning and end of the the Na + channels begin to close, leading to a decrease< pulse, it can be easily isolated by visual inspection, leav- inward current. The process by which Na + channe] ing the pure INa. The full range of current flow through close during a maintained depolarization is tenne the voltage-gated Na + channels (INa)is measured by re- inactivation. In contrast, the K+ channels in the squi peating this analysis after stepping V m to many differ- axon do not inactivate; they remain open as long as th ent levels. With a similar process, IK can be measured membrane is depolarized (Figure 9-7). when the Na + channels are blocked by tetrodotoxin Thus, depolarization causes Na + channels t (Figure 9-3C). undergo transitions among three different states,whic represent three different conformations of the Na channel protein: resting, activated, or inactivated. Upo Voltage-Gated Sodium and Potassium Conductances depolarization the channel goes from the restin Are Calculated From Their Currents (closed) state to the activated (open) state (see Figw The Na+ and K+ currents depend on two factors: the 6-6C). If the depolarization is brief, the channels go d conductance for each ion and the electrochemical rectly back to the resting state upon repolariz~tion. : driving force acting on the ion. Since the Na + and K+ the depolarization is maintained, the channels go fror membrane conductance is directly proportional to the the open to the inactivated (closed) state. Once the char number of open Na + and K+ channels, we can gain in- nel is inactivated it cannot be opened by further dep< sight into how membrane voltage controls channel larization. The inactivation can be reversed only by n opening by calculating the amplitudes and time courses polarizing the membrane to its negative restin of the Na + and K+ conductance changes in response to potential, which allows the channel to switch from th voltage-clamp (Box 9-2). inactivated to the resting state. This switch takes som Measurements of Na + and K+ conductancesat var- time because channels leave the inactivated state reI. ious levels of membrane potential reveal two functional tively slowly (Figure 9-8). similarities and two differences between the Na + and These variable, time-dependent effects of depolaJ K+ channels.Both types of channels open in responseto ization on gNaare determined by the kinetics of the gal depolarizing steps of membrane potential. Moreover, as ing reactions that control the Na + channels. Each Na the size of the depolarization increases,the probability channel has two kinds of gates that must be opene and rate of opening increasefor both types of channels. simultaneously for the channel to conduct Na + ions. A TheNa + and K+ channels differ, however, in their rates activation gate is closed when the membrane is at it of opening and in their responsesto prolonged depolar- negative and is rapidly opened by dE

~ Chapter 9 / PropagatedSignaling: The Action Potential 155

polarization; an inactivationgate is open at the resting po- The Action Potential Can Be Reconstructed From tential and closes slowly in response to depolarization. the Properties of Sodium and Potassium Channels The channel conducts only for the brief period during a depolarization when both gatesare open. Hodgkin and Huxley were able to fit their measure- reverses the two processes,closing the activation gate ments of membrane conductance changesto a set of em- rapidly and opening the inactivation gate more slowly. pirical equations that completely describe variations in After the channel has returned to the resting state, it can membraneNa + and K+ conductancesas functions of again be activated by depolarization (Figure 9-9). membrane potential and time. Using these equations 156 Part n / Cell and Molecular Biology of the Neuron

(mY) and measured values for the passive properties of the axon, they computed the expected shape and the con- duction velocity of the propagated action potential. The V. .w~~ I calculated waveform of the action potential matched the waveform recorded in the undamped axon almost per- -60 I I fectly! This dose agreement indicates that the voltage I I and time dependence of the active Na + and K+ chan- I I : +20 mV I nels, calculated from the voltage-clamp data, accurately I I I OmV, describe the properties of the channels that are essential I ~: -20 mV I for generating and propagating the action potential. A : -40 mV I half century later, the Hodgkin-Huxley model stands as I I the most successful quantitative computational model I I in neural scienceif not in all of biology. I I According to the Hodgkin-Huxley model, an action I I potential involves the following sequenceof events. A I depolarization of the membrane causesNa + channels to I I I open rapidly (an increasein gNa),resulting in an inward ~ I Na + current. This current, by discharging the mem- , I I brane capacitance,causes further depolarization, there-

Ii Time- 1 ms

Figure 9-6 Voltage-clamp experiments show that Na+ chan- nels turn on and off more rapidly than K+ channels over a wide range of membrane potentials. The increasesand de- creases in the Na+ and K+ conductances(9Na and 9\<)shown ~ here reflect the shifting of thousands of voltage-gatedchannels betweenthe open andclosed states.

Vm P, P2

Vm

Time- Figure 9-8 Sodium channels remain inactivated for a few O.5ms milliseconds after the end of a depolarization. Therefore if the interval between two depolarizingpulses (P, and P2)is Figure 9.7 Sodium and potassium channels respond differ- brief, the second pulse produces a smaller increase in 9'N.be- ently to long-term depolarization. If the membrane is repolar- cause many of the Na+ channelsare inactivated.The longer ized after a brief depolarization(line a), both 9/118and 9K return the interval between pulses, the greater the increasein 9'N., to their initial values. If depolarizationis maintained (line b), the becausea greater fraction of channelswill have recoveredfrom Na+ channelsclose (or inactivate)before the depolarizationis inactivationand returned to the resting state when the second terminated. whereas the K+ channels remain open and gK in- pulse begins. The time course of recovery from inactivation creasesthroughout the depolarization. contributes to the time course of the refractory period. Chapter 9 / PropagatedSignaling: The Action Potential 157

1 Resting(closed) 2 Activated(open) Na.

Fast channel opening .

Figure 9-9 Voltage-gated Na + channelshave + + + + + + two gates, which respond in opposite ways to depolarization. In the resting (closed)state the activation gate is closed and the inactiva- tion gate is open (1). Upon depolarizationa '-.JSIow rapid opening of the activation gate allows Na+ to flow through the channel (2). As the inactiva- tion gates close, the Na+ channelsenter the in- activated (closed)state (3). Upon repolarization, 3 Inactivated (closed) first the activation gate closes, then the inacti- vation gate opens as the channel returns to the resting state (1).

by opening more Na+ channels, resulting in a further In most cells the action potential is followed increase in inward current. This regenerative process by a transient hyperpolarization, the after-potential.This drives the membrane potential toward ENa,causing the brief increasein membrane potential occurs becausethe rising phase of the action potentiaL} The depolarizing K+ channels that open during the later phase of the ac- state of the action potential then limits the duration of tion potential closesome time after Vm hasreturned to the action potential in two ways: (1) It gradually inacti- its resting value. It takes a few milliseconds for all of the vatesthe Na+ channels,thus reducing gNa,and (2) it voltage-gated K+ channels to return to the closed state. opens, with some delay, the voltage-gated K+ channels, During this time, when the permeability of the mem- thereby increasing gK' Consequently, the inward Na + brane to K+ is greater than during the resting state, Vm current is followed by an outward K+ current that tends is hyperpolarized slightly with respect to its normal to repolarize the membrane (Figure 9-10). resting value, resulting in a Vm closer to EK (Figure 9-10). 1. It may at first seemparadoxical that to depolarizethe cell exper- The action potential is also followed by a brief pe- imentallyone passesoutward current acrossthe membrane(see Fig- riod of diminished excitability, or refractoriness, which ure 7-2C),while at the same time attributing the depolarization can be divided into two phases. The absoluterefractory during the upstroke of the action potential to an inwardNa+ cur- periodcomes immediately after the action potential; dur- rent. However, in both casesthe current flow acrossthe passive ing this period it is impossible to excite the cell no mat- components,the nongatedleakage channels (gt) and the capaci- tanceof the membrane(Cm), is outward becausepositive chargeis ter how great a stimulating current is applied. This injectedinto the cell in one casethrough an intracellular electrode phase is followed directly by the relativerefractory period, (seeFigure 7-2) and in the other caseby the opening of voltage- during which it is possible to trigger an action potential gatedNa + channels.It is a matter of conventionthat when we refer but only by applying stimuli that are stronger than to current injectedthrough a microelectrodewe refer to the direc- those normally required to reach threshold. These peri- tion in which the current crossesthe membranecapacitance and leakagechannels, whereas when we refer to current that flows ods of refractoriness, which together last just a few through channelswe refer to the direction of movementof charge milliseconds, are caused by the residual inactivation of Na+ channelsand increasedopening of K+ channels. through the channels. 158 Part n / Cell and Molecular Biology of the Neuron

ionic current (lNa + IK + II) just changes from outward to inward, depositing a net positive charge on the inside of the membrane capacitance,is the threshold.

Variations in the Properties of Voltage-Gated Ion Channels Increase the Signaling Capabilities of Neurons

The basic mechanism of electrical excitability identified by Hodgkin and Huxley in the - whereby voltage-gated ion channels conduct an inward ionic current followed by an outward ionic current-ap- pears to be universal in all excitable cells. However, Figure 9.10 The sequentialopening of voltage-gatedNa+ and K+ channels generates the action potential. One of dozens of different types of voltage-gated ion channels Hodgkin and Huxley's great achievements was to separatethe have been identified in other nerve and muscle cells, total conductancechange during an action potential. first de- and the distribution of specific types varies not only tected by Cole and Curtis (see Figure 9-1) into separate compo- from cell to cell but also from region to region within a nents attributableto the opening of Na+ and K+ channels.The cell. These differences in the pattern of ex- shape of the action potential and the underlying conductance changes can be calculatedfrom the properties of the voltage- pression have important consequencesfor the details of gated Na+ and K+ channels. membrane excitability, as we shall now explore.

The Expresses a Rich Variety of Voltage-Gated Ion Channels Although the voltage-gatedNa + and K+ channelsin the Another feature of the action potential predicted by the Hodgkin-Huxley model is its all-or-none behavior. squid axon described by Hodgkin and Huxley have A fraction of a millivolt can be the difference between a been found in almost every type of neuron examined, subthreshold depolarizing stimulus and a stimulus that several other kinds of channels have also been identi- generatesan action potential. This all-or-none phenom- fied. For example, most neurons contain voltage-gated enon may seem surprising when one considers that the Ca2+ channels that open in response to membrane de- Na+ conductanceincreases in a strictly gradedmanner polarization. A strong drives as depolarization is increased (see Figure 9-6). Each in- Ca2+ into the cell, so these channels give rise to an in- crement of depolarization increasesthe number of volt- ward lea. Some neurons and muscle cells also have volt- age-gatedNa + channelsthat switch from the closedto age-gated CI- channels. Finally, many neurons have the open state, thereby causing a gradual increase in monovalent cation-permeable channels that are slowly Na + influx. Why then is there an abrupt threshold for activated by hyperpolarization and are permeable to generating an action potential? both K+ and Na+. The net effect of the mixed perme- Although a small subthreshold depolarization in- ability of these rather nonselective channels, called creases the inward INa, it also increases two outward h-type,is the generation of an inward, depolarizing cur- currents, IK and Iv by increasing the electrochemical rent in the voltage range around the resting potential. driving force on K+ and Cl-. In addition, the depolar- Each basic type of ion channel has many variants. ization augments the K+ conductance, gKt by gradually For example, there are four major types of voltage- opening more voltage-gated K+ channels (see Figure activated K+ channels that differ in their kinetics of acti- 9-6). As IK and II increasewith depolarization, they tend vation, voltage activation range, and sensitivity to vari- to resist the depolarizing action of the Na + influx. How- ous ligands. These variants are particularly common in ever, becauseof the great voltage sensitivity and rapid the nervous system. (1) The slowly activating channel kineticsof activationof the Na+ channels,the depolar- described by Hodgkin and Huxley is called the delayed ization eventually reachesa point where the increase in rectifier.(2) A calcium-activatedK+ channelis activated by inward INa exceedsthe increasein outward IK and II' At intracellular Ca2+, but its sensitivity to intracellular this point there is a net inward current producing a fur- Ca2+ is enhanced by depolarization. It requires both a ther depolarization so that the depolarization becomes rise in internal Ca2+ (mediated by voltage-gated Ca2+ regenerative.The specificvalue of Vm at which the net channels) and depolarization to achieve a maximum Chapter 9 / PropagatedSignaling: The Action Potential 159

probability of opening. (3) The A-type K+ channelis acti- tive protein phosphatase, calcineurin, which dephos- vated rapidly by depolarization, almost as rapidly as phorylates the channel, thereby inactivating it (see Fig- the Na + channel; like the Na + channel, it also inacti- ure 6-7C). vates rapidly if the depolarization is maintained. (4) The Thus, in some cells the Ca2+influx during an action M-type K+ channelis very slowly activated by small de- potential can have two opposing effects:(1) The positive polarizations from the resting potential. One distinctive charge that it carries into the cell contributes to the re- feature of the M-type channels is that they can be closed generative depolarization, while (2) the increasein cyto- by a , (ACh). plasmic Ca2+ concentration results in the opening of Similarly, there are at least five subtypes of voltage- more K+ channels and the closing of Ca2+channels. Be- gated Ca2+ channels and two or more types of voltage- cause of the opening of K+ channels and the closing of gated Na + channels. Moreover, each of these subtypes Ca2+ channels, outward ionic current increases while has several structurally and functionally different iso- inward ionic current decreases;the resulting net efflux forms. of positive charge causes the cell to repolarize. In this The squid axon can generate an action potential way the depolarizing influx of Ca2+ through voltage- with just two types of voltage-gated channels.Why then gated Ca2+ channels is self-limited by two processes are so many different types of voltage-gated ion chan- that aid repolarization: an increase in K+ efflux and a nels found in the nervous system? The answer is that decreasein Ca2+influx. neurons with an expanded set of voltage-gated channels Calcium's role in modulating the gating of ion have much more complex information-processing abili- channels is the simplest example of a variety of second- ties than those with only two types of channels. Some messengersystems that control channel activity. Gating ways in which this large variety of voltage-gated chan- of ion channels can also be modulated by changesin the nels influences neuronal function are described below. cytoplasmic level of small organic second-messenger compounds as a result of synaptic input from other neu- rons. The gating properties of several voltage-gated Gating of Voltage-Sensitive Ion Channels Can channels that are directly involved in generating action Be Influenced by Various Cytoplasmic Factors potentials are modified when their phosphorylation In a typical neuron the opening and closing of certain state is changed by a protein kinase (eg, the cAMP- voltage-gated ion channels can be modulated by vari- dependent protein kinase) whose activity is controlled ous cytoplasmic factors, resulting in increasedflexibility by changes in the concentration of synaptically acti- of the neuron's excitability properties. Changes in such vated second messengers(eg, cAMP). The importance cytoplasmic modulator substancesmay result from the of Ca2+ and other second messengersin the control of normal intrinsic activity of the neuron itself or from the neuronal activity will become evident in many contexts influences of other neurons. throughout this book. The flow of ionic current through membrane chan- nels during an action potential generally does not result Excitability Properties Vary Between Regions in appreciable changes in the intracellular concentra- of the Neuron tions of most ion species.Calcium is a notable exception to this rule. Changesin the intracellular concentration of Different regions of the cell perform specific signaling Ca2+ can have important modulatory influences on the tasks. The axon, for example, usually specializesin car- gating of various channels. The concentration of free rying signals faithfully over long distances. As such, it ea2+ in the cytoplasm of a resting cell is extremely low, functions as a relatively simple relay line. In contrast, about 10-7 M, several orders of magnitude below the the input, integrative, and output regions of a neuron external Ca2+concentration. For this reason the intracel- (see Figure 2-8) typically perform more complex pro- lular Ca2+ concentration may increase significantly as a cessing of the information they receive before passing it result of inward current flow through voltage-gated along. The signaling function of a discrete region of the Ca2+channels. neuron depends on the particular set of ion channels The transient increase in Ca2+ concentration near that it expresses. the inside of the membrane has several effects. It en- In many types of neurons the have hancesthe probability that Ca2+-activated K+ channels voltage-gated ion channels, including ea2+, K+, and in will open. Some Ca2+ channels are themselves sensitive some casesNa + channels. When activated, these chan- to levels of intracellular Ca2+ and are inactivated when nels modify the passive, electrotonic conduction of incoming Ca2+ binds to their intracellular surface. In synaptic potentials. In some neurons action potentials other channels the influx of Ca2+ activates a Ca2+-sensi- may be propagated from their site of initiation at the 160 Part II / Cell and Molecular Biology of the Neuron

trigger zone back into the dendrites, thereby influencing with a constant-frequency train of action potentials, and synaptic integration in the dendrites. In other neurons still others with either an accelerating or decelerating the density of dendritic voltage-gated channels may train of action potentials. Some neurons even fire spon- even support the orthograde propagation of a dendritic taneously in the absenceof any external input because impulse to the cell and . of the presenceof h-type channels that generateendoge- The trigger zone of the neuron has the lowest nous pacemaker currents (Figure 9-11). threshold for action potential generation, in part be- In certain neurons small changes in the strength of cause it has an exceptionally high density of voltage- synaptic inputs produce a large increase in firing rate, gated Na+ channels. In addition, it typically has whereas in other cells large changes in synaptic input voltage-gated ion channels that are sensitive to rela- are required to modulate the firing rate. In many neu- tively small deviations from resting potential. These rons a steady hyperpolarizing input makes the cell less channels are important in determining whether synap- responsive to excitatory input by reducing the resting tic input will drive the membrane potential to spike inactivation of the A-type K+ channels. In other neurons threshold. They thus playa critical role in the transfor- sucha steadyhyperpolarization makes the cell moreex- mation of graded, analog changes in synaptic or recep- citable, becauseit removes the inactivation of a particu- tor potentials into a temporally patterned, digital train lar class of voltage-gated Ca2+ channels. In many cases of all-or-none action potentials. Examples include the the firing properties of a neuron can be modulated by M-type and certain A-type K+ channels, the hyperpolar- second messenger-mediated changes in the function of ization-activated h-~ channels, and a class of low voltage-gated ion channels (Figure 9-11). voltage-activatedCa + channels (seebelow). As the action potential is carried down the axon it is mediatedprimarily by voltage-gatedNa + and K+ chan- The Signaling Functions of Voltage-Gated nels that function much like those in the squid axon. At Channels Can Be Related to Their the nodes of Ranvier of myelinated the mecha- Molecular Structures nism of action potential repolarization is particularly simple-the spike is terminated by fast inactivation of The empirical equations derived by Hodgkin and Na+ channelscombined with a large outward leakage Huxley are quite successfulin describing how the flow of ions through the Na + and K+ channels generatesthe current. Voltage-gated K+ channels do not playa signif- icant role in action potential repolarization at the nodal action potential. However, these equations describe the membrane. process of excitation primarily in terms of changes in Presynaptic nerve terminals at chemical membrane conductance and current flow. They tell lit- commonly have a high density of voltage-gated Ca2+ tie about the molecular structure of the voltage-gated channels. Arrival of an action potential in the terminal channels and the molecular mechanisms by which they opens these channels, causing ea2+ influx, which in are activated. Fortunately, technical advances such as turn triggers transmitter release. those described in Chapter 6 have made it possible to examine the structure and function of the voltage- gated Na+, K+, and Ca2+ channels in detail at the mol- Excitability Properties Vary Among Neurons ecular level. One of the first clues that Na + channels are distinct The computing power of an entire neural circuit is en- hanced when cells in the circuit represent a wide range physical entities came from studies that measured the of functional properties, because specific functions binding of radiolabeled tetrodotoxin to nerve mem- branes. The density of voltage-gated Na + channels in within the circuit can be assigned to cells with the most appropriate dynamic properties. Thus, while the func- different was estimated by measuring the total tion of a neuron is determined to a great extent by its amount of tritium-labeled tetrodotoxin bound when anatomical relationships to other neurons (its inputs specific axonal binding sites are saturated. In nonmyeli- and its outputs), the biophysical properties of the cell nated axons the density of channels is quite low, rang- ing from 35 to 500 Na + channels per square micrometer also playa critical role. How a neuron responds to synaptic input is deter- of axon membrane in different cell types. In myelinated axons, where the Na + channels are concentrated at the mined by the proportions of different types of voltage- gated channels in the cell's integrative and trigger nodes of Ranvier, the density is much higher-between zones. Cells with different combinations of channels re- 1000and 2000 channels per square micrometer of nodal membrane. The greater the density of Na + channels in spond to a constant excitatory input differently. Some cells respond with only a single action potential, others the membraneof an axon,the greaterthe velocityat Chapter 9 / PropagatedSignaling: The Action Potential 161

A Delayed firing B Potential-dependent C neuron excitability 1

vm~ -55 mV --r ,--

\--.

2 2 D Spike accommodation

Delay" Vm -75 mV -oJ I.-. 10.6 nA I

I-:-:---i 5Oms

Figure 9.11 Repetitive firing properties vary widely among rons can fire spontaneously in brief bursts of action potentials. different types of neurons because the neurons differ in the These endogenously generated bursts are produced by current types of voltage-gated ion channels they express. flow through two types of voltage-gated ion channels. The A. Injection of a depolarizing current pulse into a neuron from gradual depolarization that leads to a burst is driven by inward the nucleus tractus solitarius normally triggers an immediate current flowing through the h-type channels, whose activation train of action potentials (1). If the cell is first held at a hyper- gates have the unusual property of opening in response to hy- polarized membrane potential, the depolarizing pulse triggers perpolarizing voltage steps. The burst is triggered by inward a spike train after a delay (2). The delay is caused by the Ca2+ current through voltage-gated Ca2+ channels that are acti- A-type K+ channels, which are activated by depolarizing synap- vated at relatively low levels of depolarization. This ea2+ influx generates sufficient depolarization to reach threshold and drive tic input. The opening of these channels generates a transient a train of Na +-dependent action potentials. The strong depolar- outward K+ current that briefly drives Vm away from threshold. These channels typically are inactivated at the resting potential ization during the burst causes the h-type channels to close and (-55 mY), but steady hyperpolarization removes the inactiva- inactivates the Ca2+ channels, allowing the interburst hyperpo- tion, allowing the channels to be activated by depolarization. larization to develop. This hyperpolarization then opens the (From Dekin and Getting 1987.) h-type channels, initiating the next cycle in the rhythm. (From McCormick and Huguenard 1992.) B. When a small depolarizingcurrent pulse is injected into a thalamic neuron at rest. only an electrotonic, subthreshold de- D. The firing properties of sympathetic neurons in autonomic polarizationis generated (1). If the cell is held at a hyperpolar- ganglia are regulated by a neurotransmitter. A prolonged ized level, the same current pulse triggers a burst of action depolarizing current normally results in only a single action potentials (2). The effectiveness of the current pulse is en- potential. This is because depolarization turns on a slowly acti- hancedbecause the hyperpolarizationcauses a type of voltage- vated K+ current, the M current. The slow activation kinetics of gated Ca2+channel to recover from inactivation.The dashed the M-type channels allow the cell to fire one action potential line indicatesthe level of the resting potential. (From Llinas and before the efflux of K+ through the M-type channels becomes Jahnsen 1982.) sufficient to shift the membrane to more negative and The data in A and B demonstrate that steady hyperpolariza- prevent the cell from firing more action potentials (a process tion, such as might be produced by inhibitory synaptic input to termed accommodation).The neurotransmitter acetylcholine a neuron, can profoundly affect the spike train pattern that a (ACh) closes the M-type channels, allowing the cell to fire neuron generates.This effect varies greatly among cell types. manyaction potentials in response to the same stimulus. (From Jones and Adams 1987.) C. In the absence of synaptic input, thalamocorticalrelay neu-

which the axon conducts action potentials. A higher Opening of Voltage-Gated ChanneIs Is All-or-None density of voltage-gated Na + channelsallows more cur- rent to flow through the excited membrane and along The current flow through a single channel cannot be the axon core, thus rapidly discharging the capacitance measured in ordinary voltage-damp experiments for of the unexcited membrane downstream (see Figure two reasons.First, the voltage clamp acts on a large area 8-6). of membrane in which thousands of channels are open- 162 Part II / Cell and Molecular Biology of the Neuron

ing and closing randomly. Second, the background A noise caused by the flow of current through passive membrane channels is much larger than the flow of cur- rent through anyone channel. Both these problems can be circumvented by electrically isolating a tiny piece of membrane in a patch-clamp electrode (seeBox 6-1). Patch-clamp experiments demonstrate that voltage- gated channels generally have only two conductance states,open and closed. Each channel opens in an all-or- none fashion and, when open, permits a pulse of current to flow with a variable duration but constant amplitude (Figure 9-12).The conductancesof single voltage-gated Na +, K+, and Ca2+ channels in the open state typically range from 1 to 20 pS, depending on channel type. One class of ea2+-activated K+ channels has an unusually B large conductanceof about 200 pS.

Redistribution of Charges Within Voltage-Gated Ip Sodium Channels Controls Channel Gating In their original study of the squid axon, Hodgkin and Huxley suggestedthat a voltage-gated channel has a net charge, the gating charge,somewhere within its wall. They postulated that a change in membrane potential causesthis charged structure to move within the plane of the membrane, resulting in a conformational change that causes the channel to open or close. They further predicted that such a charge movement would be mea- surable. For example, when the membrane is depolar- ized a positive gating charge would move from near the inner surface toward the outer surface of the membrane, owing to its interaction with the membrane electric field. Such a displacement of positive charge would re- Figure 9-12 Individual voltage-gated channels open in an duce the net separation of charge across the membrane all-or-none fashion. and hencetend to hyperpolarize the membrane. To keep A. A small patch of membrane containing only a single the membrane potential constant in a voltage-clamp ex- voltage-gatedNa + channel is electrically isolated from the rest of the cell by the patch electrode. The Na+ current that enters periment, a small extra component of outward capaci- tive current, called gating current, would have to be gen- the cell through these channelsis recorded by a current moni- erated by the voltage clamp. When the membrane tor connected to the patch electrode. current was examined by means of very sensitive tech- B. Recordings of single Na+ channels in cultured muscle cells of rats. 1. lime course of a 10 mV depolarizing voltage step ap- niques, the predicted gating current was found to flow plied across the patch of membrane (Vp = potential difference at the beginning and end of a depolarizing voltage- across the patch). 2. The sum of the inward current through the clamp step prior to the opening or closing of the Na + Na+ channels in the patch during 300 trials Up = current channels (Figure 9-13). through the patch of membrane). The trace was obtained by Analysis of the gating current reveals that activation blocking the K+ channels with tetraethylammonium and sub- and inactivationof Na+ channelsare coupled processes. tracting the capacitive current electronically. 3. Nine individual trials from the set of 300. showing six individual Na+ channel During a short depolarizing pulse net outward move- openings (circles). These data demonstrate that the total Na+ ment of gating charge within the membrane at the begin- current recorded in a conventional voltage-clamp record (see Figure 9-3C) can be accounted for by the all-or-none opening ning of the pulse is balanced by an opposite inward and closing of individualNa + channels. (From Sigworth and movement of gating chargeat the end of the pulse. How- ever, if the pulse lasts long enough for Na + inactivation Neher 1980.) to take place, the movement of gating chargeback across the membrane at the end of the pulse is delayed. The gat- Chapter 9 / PropagatedSignaling: The Action Potential 163

Figure 9.13 Gating currents directly measure the changes in charge distrib- ution associatedwith Na+ channel acti- A vation. '8 A. When the membrane is depolarized the Na+ current (lNa) first activates and then inactivates. The activation of the 'No Na+ current is preceded by a brief out- ward gating current (/g), reflecting the out- ward movement of positive charge within the Na+ channel protein associated with the opening of the activation gate. To de- tect the small gating current it is neces- sary to block the flow of ionic current B 1 Resting (closed) 2 Open 3 Inactivated (closed) through the Na+ and K+ channels and mathematically subtract the capacitive current due to charging of the lipid bi- +++ ------layer. B. Illustration of the position of the activa- tion and inactivation gates when the channel is at rest (1), when the Na+ chan- - -- +++ +++ +++ +++ nels have been opened (2), and when the Cytoplasmic channels have been inactivated (3). It is side / the movement of the positive charge on Activation Inactivation the activation gate through the mem- gate gate brane electric field that generates the gat- ing current.

ing charge is thus temporarily immobilized; only as the learned in Chapter 6, the channel behavesas if it contains Na + channelsrecover from inactivation is the charge free a filter or recognition site that selectspartly on the basis to move back acrossthe membrane. This chargeimmobi- of size, thus acting as a molecular sieve (seeFigure 6-3). lization indicates that the gating charge cannot move The easewith which ions with good hydrogen-bonding while the channel is in the inactivated state, ie, while the characteristics pass through the channel suggests that inactivation gate is closed (seeFigure 9-9). part of the inner wall of the channel is made up of nega- To explain this phenomenon, Clay Armstrong and tively polarized or charged amino acid residues that can Francisco Bezanilla proposed that Na + channel inacti- substitute for water. When the pH of the fluid surround- vation occurs when the open (activated) channel is ing the cell is lowered, the conductanceof the open chan- blocked by a tethered plug (the ball and chain mecha- nel is gradually reduced, consistent with the titration of nism), thereby preventing the closure of the activation important negatively charged carboxylic acid residues. gate. In support of this idea, exposing the inside of the The selectivity filter of the Na + channel is made up axon to proteolytic enzymes selectively removes inacti- of four loops within the molecule (the P region) that are vation, causing the Na + channels to remain open during similar in structure (seebelow). A residue a depolarization, presumably becausethe enzymes clip is situated at equivalent points in two of these loops. A off the inactivation "ball." lysine and an alanine residue are situated at the equiva- lent site in the other two loops. The channel is thought to select for Na + ions by the following mechanism. The The Voltage-Gated Selects for negatively charged carboxylic acid groups of the glu- Sodium on the Basis of Size, Charge, and Energy tamic acid residues,which are located at the outer mouth of Hydration of the Ion of the pore, perform the first step in the selection process After the gatesof the Na+ channel have opened, how by attracting cations and repelling anions. The cations does the channel discriminate between Na + and other then encounter a constricted portion of the pore, the se- ions?The channel's selectivity mechanism can be probed lectivity filter, with rectangular dimensions of by measuring the channel's relative permeability to sev- 0.3 x 0.5 nm. This crosssection is just large enough to ac- eral typesof organicand inorganiccations that differ in commodate one Na + ion contacting one water molecule. size and hydrogen-bonding characteristics. As we Cations that are larger in diameter cannot pass through 164 Part n / Cell and Molecular Biology of the Neuron

Na+ channel.First, the a-subunit is composedof four the pore. Cations smaller than this critical size pass through the pore, but only after losing most of the waters internal repeats (domains I-M, with only slight varia- of hydration they normally carryin freesolution. tions, of a sequence that is approximately 150 amino The negative carboxylic acid group, as well as other adds in length. Each of the four repeats of this sequence oxygen atoms that line the pore, can substitute for these domain is believed to have six membrane-spanning hy- waters of hydration, but the degree of effectiveness of drophobic regions (51-56) that are primarily a-helical in this substitution varies among ion species.The more ef- form. A seventh hydrophobic region the P region that fective the substitution, the more readily ions can tra- connectsthe S5 and 56 segments,appears to form a lOOp versethe Na+ channel.The Na+ channelexcludes K+ that dips into and out of the membrane (Figure 9-14). ions, in part becausethe larger-diameter K+ ion cannot The four repeated domains are thought to be arranged interact as effectively with the negative carboxylic roughly symmetrically, with the P region and some of group. The lysine and alanine residues also contribute the membrane-spanning regions forming the walls of to the selectivity of the channel. When these residues are the water-filled pore (Figure 9-15). The second structural feature of the Na + channelre- changed to glutamic acid residues by site-directed mu- tagenesis,the Na+ channelscan act as Ca2+-selective vealed by amino acid sequenceanalysis is that one of the six putative membrane-spanning regions, the 54 re- channels! (The mechanism whereby K+ selectivity is gion, is structurally quite similar in the Na + channels of achieved was discussed in Chapter 6.) many different species.This strict conservation suggests that the 54 region is critical to Na + channel function. Moreover, the 54 region of the Na + channel is similar to Genes Encoding the Potassium,Sodium, and Calcium Channels Stem From corresponding regions of the voltage-gated Ca2+ and a Common Ancestor K+ channels (Figure 9-14) but is lacking in K+ chapnels that are not activated by voltage (see below). For this Since a change in two amino acid residues can cause a reason the 54 region may be the voltage sensor-that Na+ channelto behaveas a Ca2+channel, it is reason- part of the protein that transduces depolarization of the able to believethat the Na+ and CaB channelsmay be cell membrane into a gating transition within the chan- closely related. Detailed molecular studies have re- nel, thereby opening the channel. This idea is supported vealed that all voltage-gated ion channels-those for by the observation that the 54 region contains a distinc- tive pattern of amino acids. Every third amino acid K+, Na +, and ea2+-share several functionally impor- tant domains and are indeed quite similar. In fact, there along the 54 helix is positively charged (lysine or argi- is now strong evidence from studies of bacteria, , nine) while the intervening two amino acids are hydro- , and that most voltage-sensi- phobic. This highly charged structure is therefore likely tive cation channels stem from a common ancestral to be quite sensitive to changes in the electric field channel-perhaps a K+ channel-that can be traced to a across the membrane. Experiments using site-directed single-cell organism living over 1.4 billion years ago, be- mutagenesis show that reducing the net positive charge fore the evolution of separate and king- in one of the 54 regions of the channel lowers the volt- age sensitivity of Na + channelactivation. doms. The amino acid sequences conserved through evolution help identify the domains within contempo- Structure-function studies based on genetic engi- rary cation channels that are critical for function. neering of the a-subunit have led to a hypothesis about Molecular studies of the voltage-sensitive cation how the chargesin the 54 region move acrossthe mem- channelsbegan with the identification of Na+ channel brane during channel gating. According to the scheme, molecules. Three subunits have been isolated: one large at rest one of the charged residues on the 54 a-helix is glycoprotein (a) and two smaller polypeptides (~1 and completely buried in the wall of the channel, where its ~2). The a-subunit is ubiquitous, and insertion of this positive charge is stabilized by interaction with a nega- subunit into an artificial reconstitutes the tively charged amino acid residue in one of the other basicfeatures of Na+ channelfunction. Thereforethe a- membrane-spanning segments of the channel (Figure subunit is presumed to form. the aqueous pore of the 9-16). The other positive charges are located on parts of channel. The smaller subunits, whose presencevaries in the 54 helix that are within a water-filled lacuna in the different regions of the nervous system, regulate vari- wall of the channel that is continuous with the cyto- ous aspectsof a-subunit function. plasm. When the membrane is depolarized the change Examination of the amino acid sequence encoded in electrostatic force causes movement of the 54 helix by the clonedgene for the a-subunitof the Na+ channel relative to the surrounding channel wall, translocating reveals two fundamental features of the structure of the some of the positively charged residues to the outside of Chapter 9 / PropagatedSignaling: The Action Potential 165

Na+ channel II III IV Extracellular side

Figure 9-14 The pore-forming subunits of the voltage-gated Na+, Ca2+,and K+ channels are composed of a common re- peated domain. The a-subunit of the Na+ andCa2+ channels consists of a single polypeptide chain with four repeats (I-IV)of a domain that contains six mem- Ca2+channel brane-spanninga-helical regions (S1-S6).A stretch of amino acids, the P region between a-helices 5 and 6, forms a loop that dips into and out of the membrane.The S4 segment is shown in red, rep- resenting its net positive charge. The fourfold repetition of the P region is believed to form a ma- jor part of the pore lining (see Figure9-15). The K+ channel, in contrast. has only a single repeat of the six a-helices and the P re- gion. Four K+ channel subunits are assembledto form a com- plete channel (see Figure 6-12). (Adaptedfrom Catterall 1988, K+ channel Stevens 1991.)

domain that is repeatedfour timesin the genesfor Na+ the membrane. This movement is somehow transduced into opening of the activation gate. and Ca2+ channels. Nevertheless, the basic channel The genesencoding the major a-subunits of several structure is similar for the three channel types, as four voltage-gated Ca2+ channels have also been cloned. a-subunits must aggregatesymmetrically around a cen- Their sequencesreveal that the Ca2+ channels are also tral pore to form a K+ channel. It is this striking homol- composed of four repeating domains, each with six ogy among the voltage-gated Na +, Ca2+, and K+ hydrophobic transmembrane regions and one P loop, channels that suggests that all three channels belong to which have amino acid sequenceshomologous to those the same gene family and have evolved by gene dupli- of the voltage-gatedNa + channel(see Figure 9-14). cation and modification from a common ancestralstruc- The K+ channel genes contain only one copy of the ture, presumably a K+ channel. 166 Part n / Cell and Molecular Biology of the Neuron

The modular design of this extended gene family is also illustrated by a comparison of activation and inacti- vation mechanisms of various channels within the family. The 54 membrane-spanning region, which is thought to be the voltage sensor in channelsof this fam- ily, hasa relativelylarge net chargein the Na+, K+, and Ca2+ channels that open in response to depolarization. In contrast, the 54 regions in cyclic nucleotide-gated channels, which are only weakly sensitive to voltage, have significantly less net charge, and h-type channels lack certain conserved 54 residues. Moreover, inward- rectifying K+ channels,which have essentially no intrin- sic voltage sensitivity, completely lack the 54 region. These inward-rectifying channels are activated by the effect of hyperpolarization on freely diffusible, posi- tively charged blocking particles in the cytoplasm. De- Figure 9-15 The four membrane-spanning domains of the pending on the subspeciesof channel, this blocker may a-subunit in voltage-gated Na+ and Ca2+ channels form the be either Mi+ or various organic polyamines. These channel pore. The tertiary structure of the channels proposed channels open when the cation-blocking particle is elec- here is based on the secondary structures shown in Figure trostatically drawn out of the channel at negative poten- 9-14. The central pore is surrounded by the four internally re- tials around the resting potential. peated domains (M-I to M-IV). (Only three of the domains are shown here for clarity.) Each quadrant of the channel includes Inactivation of voltage-gated ion channels is also six cylinders, which represent six putative membrane-spanning mediated by different molecular modules. For eXample, a-helices. The 54 segment (in red) is thought to be involved in the rapid inactivation of both the A-type K+ channel gating because it contains a significant net charge. The protrud- and the voltage-gatedNa + channelcan be attributedto ing loop in each quadrant represents the P region segment that a tethered plug that binds to the inner mouth of the dips into the membrane to form the most narrow region of the wall of the pore. channel when the activation gate opens. In the A-type K+ channel the plug is formed by the cytoplasmic N ter- minus of the channel,whereas in voltage-gatedNa + channels the cytoplasmic loop connecting domains ill and IV of the a-subunit forms the plug. The conservative mechanism by which evolution proceeds-creating new structural or functional entities Various Smaller Subunits Contribute to the by modifying, shuffling, and recombining existing gene Functional Properties of Voltage-Gated Sodium, sequences-is illustrated by the modular design of vari- Calcium, and Potassium Channels ous members of the extended gene family that includes the voltage-gated Na +, K+ I and ea2+ channels. For ex- Most, perhaps all, voltage-gated Na +, K+, and Ca2+ ample, the basic structures of both a Ca2+-activated K+ channels have ~- and, in some cases,-y- and ~bunits channel, an h-type cation channel activated by hyperpo- that modulate the functional properties of the channel- larization and intracellular cycle neucleotides, and a forming a-subunits. The modulatory function of these voltage-independent cation channel activated by intra- subunits, which may be either cytoplasmic or mem- cellular cyclic nucleotides are the same as the structures brane-spanning, depends on the type of channel. For of other members of the gene family (six membrane- example, the subunits may enhance the efficiency of spanning a-helices and a P region), with some modifica- coupling of depolarization to activation or inactivation tions. The functional differences between these two gating. They may also shift the gating functions to dif- channels are due primarily to the addition of regulatory ferent voltage ranges. In some K+ channels in which domains that bind Ca2+ or cyclic nucleotides, respec- the a-subunit lacks a tethered inactivation plug, addi- tively, to the C-terminal ends of the proteins. As we saw tion of a set of ~-subunits with their own N-terminal in Chapter 6, the subunits that comprise the inward- tethered plugs can endow the channel with the ability rectifying K+ channelsare truncated versions of the fun- to rapidly inactivate. In contrast to the a-subunits, damental domain, consisting of the P region and its two there is no known homology among the ~-, -y-, and flanking membrane-spanning regions. Four such sub- 8-subunits from the three-major subfamilies of voltage- units combine to form a functional channel (Figure 9-17). gated channels. Chapter 9 / PropagatedSignaling: The Action Potential 167

A B

+ + + +

Figure 9.16 Gating of the Na+ channel is thought to rely on cavity in the channel wall that is continuous with the cyto- redistribution of net charge in the 54 region. plasm. A. At rest, the inside-negativeelectric field across the mem- B. When the cell is depolarizedthe change in electrical field brane biases the positively charged 54 helix toward the inside across the membrane drives the S4 region toward the extra- of the membrane. One of the positive charges is stabilized by cellular face of the membrane. This change in configuration interactionwith a negative charge in another part of the chan- opens the activation gate by a mechanism that is not well nel. The remainder of the charged region lies in a water-filled understood. (Adaptedfrom Yanget al. 1996.)

The Diversity of Voltage-Gated Channel Types Is creased either by alternative splicing of the pre-mRNA Due to Several Genetic Mechanisms molecule or by the encoding of different variants of a basic ~-subunit type on different genes. These various A single ion species can cross the membrane through sources of diversity endow the nervous system with several distinct types of ion channels, each with its own tremendous opportunities for regional diversity of func- characteristic kinetics, voltage sensitivity, and sensitiv- tional properties. ity to different modulators. In voltage-gated channels this diversity may be due to any of five genetic mecha- nisms: (1) More than one gene may encode related a- Mutations in Voltage-Gated Channels Cause subunits within each class of channel. (2) A single gene Specific Neurological Diseases product may be alternatively spliced in different classes of neurons, resulting in different variants of the mRNA Several inherited neurological disorders are now known that encodesthe a-subunit. (3) The four a-subunits that to be caused by mutations in voltage-gated ion chan- coalesceto form a K+ channel may be encoded by dif- nels. Patients with hyperkalemic periodic ferent genes. After the gene products are have episodes of muscle stiffness () and mus- mixed and matched in various combinations, thus form- cle (paralysis) in response to the elevation of ing different subclassesof heteromultimeric channels. K+ levels in serum after vigorous exercise.Genetic stud- (4) A given a-subunit may be combined with different ies have shown that the diseaseis causedby a point mu- Ih -y- or 8-subunits to form functionally different chan- tation in the a-subunit of the gene for the voltage-gated nel types. (5) The diversity of some j3-subunits is in- Na+ channel found in . Voltage-damp 168 Part II / Cell and Molecular Biology of the Neuron

Extracellular side A Depolarization-activated, noninactivating K+ channel Cytoplasmic side Selectivity determinant NH2 COOH

B Depolarization-activated. inactivating K+ channel

C Der.°1arization- and Ca +-activated K+ channel

NH2

D Cyclic nucleotide- activated cationchannel

Cyclic nucleotide-binding site NH2

E Inward-rectifier K+ channel

Figure 9-17 Ion channelsbelonging to the extendedgene and intracellularCa2+ have a Ca2+-binding sequenceattached family of voltage-gatedchannels are variantsof a common to the C-terminal end of the channel. moleculardesign. D. Cation channelsgated by cyclic nucleotides have a cyclic A. Depolarization-activated.noninactivating K+ channels are nucleotide-bindingdomain attached to the C-terminalend. One formed from four copies of an a-subunit, the basic building class of such channelsis the voltage-independent,cyclic block of voltage-gatedchannels. The a-subunit is believed to nucleotide-gatedchannels important in the transductionof have six membrane-spanningregions and one membrane- olfactory and visual sensory signals. Another class of channels embedded region (the P region). The P region contains a K+- is the hyperpolarization-activatedh-type channelsimportant for selective sequence (denoted by the rectangle). pacemakeractivity (see Figure 9-11 C). B. Many K+ channels that are first activated and then inacti- E. Inward-rectifying K+ channels, which are gated by blocking vated by depolarization have a ball-and-chain segment on their particles available in the cytoplasm, areformed from truncated N-terminal ends that inactivates the channel by plugging its versions of the basic building block, with only two inner mouth. membrane-spanning regions and the P region. C. Potassiumchannels that are activated by both depolarization Chapter 9 / PropagatedSignaling: The Action Potential 169

studies of cultured skeletal muscle cells obtained from structure-about the primary amino acid sequence of biopsies of patients with this disorder demonstrate that the proteins that form them. Now these two approaches the voltage-gatedNa + channelsfail to completelyinac- are being combined in a concerted effort to understand tivate. This defect is exacerbatedby elevation of external the relationship between structure and function in these K+. The prolonged opening of the Na+ channelsis channels: how they are put together, what their con- thought to cause muscles to fire repetitive trains of ac- tours and surface map look like, how they interact with tion potentials, thus producing the muscle stiffness. As other molecules, what the structure of the channel pore the fraction of channels with altered inactivation in- is, and how its gate is opened. creases(as a result of continued K+ elevation), the mus- Thus, we may soon be able to understand the mo- cle resting potential eventually reachesa new stable de- lecular mechanism for the remarkable ability of voltage- polarized level (around -40 mY), at which point most gated channels to generate the action potential. These Na+ channelsbecome inactivated so that the membrane insights have two important implications: they will fails to generatefurther action potentials (paralysis). allow us to understand better the molecular bases of Patients with episodic ataxia exhibit normal neuro- certain genetic diseasesthat involve mutations in ion logical function except during periods of emotional or channel genes, and they will enable us to design safer physical stress, which can trigger a generalized ataxia and more effective drugs to treat a variety of diseases due to involuntary muscle movements. The diseasehas that involve disturbances in electrical signaling (such as been shown to result from one of several point muta- epilepsy, ,myotonia, and ataxia). tions in a delayed-rectifier, voltage-gated K+ channel. These mutations decreasecurrent through the channel, in part by enhancing the rate of inactivation. As a result, becauseless outward K+ current is available for repolar- JOM Koester ization, the tendency of nerves and muscle cells to fire Steven A. Siegelbaum repetitively is enhanced.(Remarkably, the first K+ chan- nel gene to be cloned was identified based on a genetic strategy involving a similar mutation in a DrosophilaK+ channel gene, which gives rise to the so-called phenotype.) Muscle diseases involving mutations in Cl- channels () and Ca2+ channels (hypokalemic periodic paralysis) have also been identi- Selected Readings fied. Armstrong CM. 1992. Voltage-dependent ion channels and their gating. Physiol Rev 72:55-13. Armstrong CM, Hille B. 1998. Voltage-gated ion channels An Overall View and electrical excitability. Neuron 20:371-380. Cannon SC. 19%. Ion-channel defects and aberrant excitabil- An action potential is produced by the movement of ity in myotonia and periodic paralysis. Trends Neurosci ions acrossthe membrane through voltage-gated chan- 19:~1O. nels. This ion movement, which occurs only when the Catterall WA. 1994.Molecular properties of a superfamily of channels are open, changes the distribution of charges plasma-membrane cation channels. CUff Opin Cell BioI on either side of the membrane. An influx of Na +, and 6:607-615. in some casesCa2+, depolarizes the membrane, initiat- Hille B. 1991. Ionic Channelsof ExcitableMembranes, 2nd ed. ing an action potential. An outflow of K+ then repolar- Sunderland, MA: Sinauer. izes the membrane by restoring the initial charge distri- Hodgkin AL. 1992. Chance& Design: Reminiscencesof Science bution. A particularly important subset of voltage-gated in Peaceand War.Cambridge: Cambridge Univ. Press. 150m LL, De Jongh KS, Catterall WA. 1994. Auxiliary sub- ion channels opens primarily when the membrane p0- units of voltage-gated ion channels. Neuron 12:1l~1194. tential nears the threshold for an action potential; these Jan LY, Jan YN. 1997. Cloned potassium channels from channels have a profound effect on the firing patterns and prokaryotes. Annu Rev Neurosci 20:91-123. generatedby a neuron. Kukuljan M, Labarca P, Latorre R. 1995. Molecular determi- We know something about how channels function nants of ion conduction and inactivation in K+ channels. from studies using variations on the voltage-clamp Am J Physiol 268:C535-C556. technique-these studies let us eavesdrop on a channel Llinas RR. 1988.The intrinsic electrophysiological properties at work. And we know something from biochemical of mammalian neurons: insights into central nervous sys- and molecular biology studies about the channel's tem function. Science242:1654-1664. 170 Part n / Cell and Molecular Biology of the Neuron

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