signaling at the nerve-muscle synapse: directly gated transmission · 2004-04-12 · recordings are...

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Signaling at the Nerve-Muscle Synapse:Directly Gated Transmission

The Neuromuscular Junction Is a Well-Studied Example ofDirectly Gated Synaptic Transmission

The Motor Neuron Excites the Muscle by Opening IonChannels at the End-Plate

The Synaptic Potential at the End-Plate Is Produced byIonic Current Flowing Through Acetylcholine-GatedChannels

The Ion Channel at the End-Plate Is Permeable to BothSodium and Potassium

The Current Flow Through Single Ion Channels Can BeMeasured by the Patch Oamp

Individual Acetylcholine-Gated Channels Conduct aUnitary Current

Four Factors Determine the End-Plate Current

The Molecular Properties of the Acetylcholine-GatedChannel at the Nerve-Muscle Synapse Are Known

Ugand-Gated Channels for Acetylcholine Differ FromVoltage-Gated Channels

A Single Macromolecule Forms the NicotinicAcetylcholine Receptor and Channel

,An Overall View

Postscript: The End-Plate Current Can Be Calculated Froman Equivalent Circuit

S YNAPTIC COMMUNICATION in the brain relies mainlyon chemical mechanisms. Before we examine thecomplexities of chemical synaptic transmission

in the brain, however, it will be helpful to examinethe basic features of chemical synaptic transmissionat the site where they were first studied and remainbest understood-the nerve-muscle synapse, the junc-

tion between a motor neuron and a skeletal musclefiber.

The nerve-muscle synapse is an ideal site for study-ing chemical signaling because it is relatively simple andalso very accessible to experimentation. The muscle cell islarge enough to accommodate the two or more microelec-trades needed to make electrical measurements. Also, thepostsynaptic muscle cell is normally innervated by justone presynaptic axon, in contrast to the convergent con-nections on central nerve cells. Most importantly, chemi-cal signaling at the nerve-muscle synapse involves a rela-tively simple mechanism. Release of neurotransmitterfrom the presynaptic nerve directly opens a single type ofion channel in the POSt-synaptic membrane.

The Neuromuscular Junction Is a Well-StudiedExample of Directly Gated SynapticTransmission

The axon of the motor neuron innervates the muscleat a specialized region of the muscle membrane calledthe end-plate (see Figure 11-1). At the region where themotor axon approaches the muscle fiber, the axon losesits myelin sheath and splits into several fine branches.The ends of the fine branches form multiple expansionsor varicosities, called synaptic boutons, from which themotor neuron releases its transmitter (Figure 11-1). Eachbouton is positioned over a junctional fold, a deep de-pression in the surface of the postsynaptic muscle fiberthat contains the transmitter receptors (Figure 11-2).The transmitter released by the axon terminal is acetyl-choline (ACh), and the receptor on the muscle mem-

Part ill / Elementary Interactions Between Neurons: Synaptic Transmission188

Figure 11-1 The neuromuscular junction isreadily visible with the light microscope. Atthe muscle the motor axon ramifies into sev-eral fine branches approximately 2 ""m thick.Each branch forms multiple swellings calledpresynaptic boutons, which are covered by athin layer of Schwann cells. The boutons lieover a specialized region of the muscle fibermembrane, the end-plste, and are separatedfrom the muscle membrane by a 100 nmsynaptic cleft. Each presynaptic bouton con-tains mitochondria and synaptic vesicles clus-tered around active zones, where the acetyl-choline (ACh) transmitter is released.Immediately under each bouton in the end-plate are several junctional folds, which con-tain a high density of ACh receptors at theircrests. The muscle fiber is covered by a layerof connective tissue, the basement mem-brane (or basal lamina), consisting of collagenand glycoproteins. Both the presynaptic ter-minal and the muscle fiber secrete proteinsinto the basement membrane, including theenzyme acetylcholinesterase, which inacti-vates the ACh released from the presynapticterminal by breaking it down into acetate andcholine. The basement membrane also orga-nizes the synapse by aligning the presynapticboutons with the postsynaptic junctionalfolds. (Adapted in part from McMahan andKuffler 1971.)

Neuromuacul8f junction

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FIgure 11-2 Electron microscope autora-diograph of the vertebrate neuromuscu-lar junction, showing localization of AChreceptors (black developed grains) at thetop one-third of the postsynaptic junc-tional folds. This receptor-rich region ischaracteriz8d by an increased density ofthe post junctional membrane (arrow). Themembrane was incubated with radiolabeleda-bungarotoxin, which binds to the ACh re-ceptor. Radioactive decay results in theemittance of a radioactive particle, causingsilver grains to become fixed (dark grains),Magnification x 18,000.

brane is the nicotinic type of ACh receptor11-3).1

The presynaptic and postsynaptic membranes areseparated by a synaptic cleft around 100 nm wide.Within the cleft is a basement membrane composed ofcollagen and other extracellular matrix proteins. Theenzyme acetylcholinesterase, which rapidly hydrolyzesACh, is anchored to the collagen fibrils of the base-ment membranes. In the muscle cell, in the region be-low the crest of the junctional fold and extending intothe fold, the membrane is rich in voltage-gated Na +

channels.Each presynaptic bouton contains all the machinery

required to release neurotransmitter. This includes thesynaptic vesicles, which contain the transmitter ACh, andthe active zone, a part of the membrane specialized forvesicular release of transmitter (see Figure 11-1). Everyactive zone in the presynaptic membrane is positionedopposite a junctional fold in the post$ynaptic cell. At thecrest of each fold the receptors for ACh are clustered in alattice, with a density of about 10,000 receptors persquare micrometer (Figures 11-2 and 11-3). In addition,each active zone contains voltage-gated ea2+ channels

1 There are two basic types of receptors for ACh: nicotinic and mU&-

ca.rinic. The nicotinic receptor is an ionotropic receptor while themUBCarinic receptor is a metabotropic receptor (see Chapter 10). Thetwo receptors can be distinguished further because certain drugsthat simulate the actions of ACh-that is, nicotine and muscarine-bind exclusively to one or the other type of ACh receptor. We shalllearn about mUBCarinic ACh receptors iri Chapter 13.

Chapter 11 / Signaling at the Nerve-Muscle Synapse: Directly Gated Transmission 189

that permit Ca2+ to enter the terminal with each actionpotential (see Figure 11-1). This influx of Ca2+ triggersfusion of the synaptic vesicles in the active zones withthe plasma membrane, and fusion leads to release of thevesicle's content into the synaptic deft.

(Figure

Receptor

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Figure 11-3 Reconstructed electron microscope image ofthe ACh receptor-channel complex in the fish Torpedo cali-fornica. The image was obtained by computer processing ofnegatively stained images of ACh receptors. The resolution is1.7 nm, fine enough to see overall structures but too coarse toresolve individual atoms. The overall diameter of the receptorand its channel is about 8.5 nm. The pore is wide at the exter-nal and internal surfaces of the membrane but narrows consid-erably within the lipid bilayer. The channel extends some dis-tance into the extracellular space. (Adapted from Toyoshimaand Unwin 1988.)

190 Part ill / Elementary Interactions Between Neurons: Synaptic Transmission

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Figure 11.4 The end-plate potential can be isolated phar-macologically for study.A. Under normal circumstances stimulation of the motor axonproduces an action potential in a skeletal muscle cell. Thedashed line shows the inferred time course of the en~latepotential that triggers the action potential.

B. The end-plate potential can be isolated in the presence ofcurare, which blocks the binding of ACh to its receptor and soprevents the end-plate potential from reaching the threshold foran action potential (dashed line). In this way the currents andchannels that contribute to the end-plate potential, which aredifferent from those producing an action potential. can be stud-ied. The values for the resting potential (-90 mY), end-platepotential, and action potential shown in these intracellularrecordings are typical of a vertebrate skeletal muscle.

The Motor Neuron Excites the Muscle byOpening Ion Channels at the End-Plate

Upon release of ACh from the motor nerve terminal, themembrane at the end-plate depolarizes rapidly. The ex-citatory postsynaptic potential in the muscle cell iscalled the end-plate potential. The amplitude of the end-plate potential is very large; stimulation of a single mo-tor cell produces a synaptic potential of about 70 m V.This change in potential usually is large enough torapidly activate the voltage-gated Na + channels in the

junctional folds. This converts the end-plate potentialinto an action potential, which propagates along themuscle fiber. (In contrast, in the central nervous systemmost presynaptic neurons produce postsynaptic poten-tials less than 1 m V in amplitude, so that input from

many presynaptic neurons is needed to generate an ac-tion potential there.)

The Synaptic Potential at the End-Plate Is Producedby Ionic Current Flowing Through Acetylcholine-Gated Channels

The end-plate potential was first studied in detail inthe 19508 by Paul Fatt and Bernard Katz using intra-cellular voltage recordings. Fatt and Katz were able toisolate the end-plate potential using the drug curare2 toreduce the amplitude of the end-plate potential belowthe threshold for the action potential (Figure 11-4). Theyfound that the synaptic potential in muscle cells waslargest at the end-plate, decreasing progressively withdistance from the end-plate region (Figure 11-5). Theyconcluded that the synaptic potential is generated by aninward ionic current confined to the end-plate region,which then spreads passively away from the end-plate.(Remember, an inward current corresponds to an influxof positive charge, which will depolarize the inside ofthe membrane.) Current flow is confined to the end-plate because the ACh-activated ion channels are local-ized there, opposite the presynaptic terminal fromwhich transmitter is released.

The synaptic potential at the end-plate rises rapidlybut decays more slowly. The rapid rise is due to the sud-den release of ACh into the synaptic cleft by an actionpotential in the presynaptic nerve terminal. Once re-leased, ACh diffuses rapidly to the receptors at the end-plate. Not all the ACh reaches postsynaptic receptors,however, because it is quickly removed from the synap-tic cleft by two processes: hydrolysis and diffusion outof the synaptic cleft.

The current that generates the end-plate potentialwas first studied in voltage-clamp experiments (see Box9-1). These studies revealed that the end-plate currentrises and decays more rapidly than the resultant end-plate potential (Figure 11-6). The time course of the end-plate current is directly determined by the rapid open-ing and closing of the ACh-gated ion channels. Becauseit takes time for an ionic current to charge or dischargethe muscle membrane capacitance, and thus alter themembrane voltage, the end-plate potential lags behindthe synaptic current (see Figure 8-3 and the Postscript atthe end of this chapter).

2 Curare is a mixture of plant toxins used by South American Indi-ans, who apply it to arrowheads to paralyze their quarry. 'I\J.~curarine, the purified active agent, blocks neuromuscular transmis-sion by binding to the nicotinic ACh receptor, preventing its activa-tion by ACh.

Figure 11-5 The synaptic potential in muscleis largest at the end-plate region and pas-sively propagates away from it. (Adapted fromMiles 1969.)A. The amplitude of the synaptic potential de-cays and the time course of the potential slowswith distance from the site of initiation in theend-plate.B. The decay results from leakiness of the mus-cle fiber membrane. Since current flow mustcomplete a circuit, the inward synaptic currentat the end-plate gives rise to a retum flow ofoutward current through resting channels andacross the membrane (the capacitor). It is thisretum flow of outward current that produces thedepolarization. Since current leaks out all alongthe membrane, the current flow decreases withdistance from the end-plate. Thus, unlike the re-generative action potential, the local depolariza-tion produced by the synaptic potential of themembrane decreases with distance.

The Ion Channel at the End-Plate Is Permeableto Both Sodium and Potassium

Why does the opening of the ACh-gated ion channelslead to an inward current flow that produces the depo-larizing end-plate potential? And which ions movethrough the ACh-gated ion channels to produce this in-ward current? One important due to the identity of theion (or ions) responsible for the synaptic current can beobtained from experiments that measure the value ofthe chemical driving force propelling ions through thechannel. Remember, from Chapter 7, the current flowthrough a membrane conductance is given by the prod-uct of the membrane conductance and the electrochemi-cal driving force on the ions conducted through thechannels. The end-plate current tha~ underlies the exci-tatory postsynaptic potential (EPSP)"is defined as

IEPSP = gEPSP X (V m - EEPSP)' (11-1)

where IEPSP is the end-plate current, gEPSP is the conduc-tance of the ACh-gated channels, V m is the membranepotential, and EEPSP is the chemical driving force, or bat-tery, that results from the concentration gradients of theions conducted through the ACh-gated channels. Thefact that current flowing through the end-plate is in-ward at the normal resting potential of a muscle cell(-90 mY) indicates that there is an inward (negative)electrochemical driving force on the ions that carry cur-rent through the ACh-gated channels at this potential.Thus, EEPSP must be positive to -90 mY.

Chapter 11 / Signaling at the Nerve-Muscle Synapse: Directly Gated 1icmsmission 191

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From Equation 11-1 we see that the value of EEPSPcan be determined by altering the membrane potentialin a voltage-clamp experiment and determining its ef-feet on the synaptic current. Depolarizing the mem-brane reduces the net inward electrochemical drivingforce, causing a decrease in the magnitude of the inwardend-plate current. If the membrane potential is set equalto the value of the battery representing the chemical dri-ving force (EEPSP), no net synaptic current will flowthrough the end-plate because the electrical drivingforce (due to V m) will exactly balance the chemical dri-ving force (due to EEPSP)' The potential at which the netionic current is zero is the reversal potential for currentflow through the synaptic channels. By determining thereversal potential we can experimentally measure thevalue of EEPSP, the chemical force driving ions throughthe ACh-gated channels at the end-plate. If the mem-brane potential is made more positive than EEPSP, therewill be a net outward driving force. In this case stimula-tion of the motor nerve leads to an outward ionic cur-rent, by opening the ACh-gated channels, and this out-ward ionic current hyperpolarizes the membrane.

If an influx of Na + were solely responsible for the

end-plate potential, the reversal potential for the excita-tory postsynaptic potential would be the same as theequilibrium potential for Na+, or +55 mY. Thus, if themembrane potential is experimentally altered from-100 to +55 mY, the end-plate current should diminishprogressively because the electrochemical driving force

192 Part m / Elementary Interactions Between Neurons: Synaptic Transmission

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Figure 11.8 The end-plate current rises and decays morerapidly than the end-plate potential.A. The membrane at the end-plate is voltag~lamped by insert-ing two microelectrodes into the muscle at the end-plate. Oneelectrode measures membrane voltage (Vm) and the secondpasses current (1m). Both electrodes are connected to a feed-back amplifier, which ensures that the proper amount of cur-rent (1m) is delivered so that Vm will be clamped at the com-mand potential Vc. The synaptic current evoked by stimulating

on Na+ (Vm - EN.> is reduced. At +55 mV the inward

current flow should be abolished, and at potentialsmore positive than +55 mV the end-plate currentshould reverse in direction and flow outward.

Instead experiments at the end-plate showed that asthe membrane potential is reduced, the inward currentrapidly becomes smaller and is abolished at 0 m V! Atvalues more positive than 0 m V the end-plate currentreverses direction and begins to flow outward (Figure11-7). This particular value of membrane potential is notequal to the equilibrium potential for Na +, or for thatmatter any of the major cations or anions. In fact, thispotential is produced not by a single ion species but bya combination of ions. The synaptic channels at the end-plate are almost equally permeable to both majorcations, Na + and K+. Thus, during the end-plate poten-tial Na + flows into the cell and K+ flow8-out. The rever-

sal potential is at 0 m V because this is a weighted aver-age of the equilibrium potentials for Na + and K+ (Box11-1). At the reversal potential the influx of Na + is bal-

anced by an equal efflux of K+.Why are the ACh-gated channels at the end-plate

not selective for a single ion species like the voltage-gated channels selective for either Na + or K+? The pore

diameter of the ACh-gated channel is thought to be sub-stantially larger than that of the voltage-gated channels.Electrophysiological measurements suggest that thepore may be up to 0.8 nm in diameter in mammals. Thisestimate is based on the size of the largest organic cationthat can permeate the channel. For example, the perme-

Tlme-

the motor nerve can then be measured at a constant mem-brane potential, for example -90 mV (see Box 9-1).B. The end-plate potential, measured when the voltage clampis not active, changes relatively slowly, following in time the in-ward synaptic current measured under voltage-clamp condi-tions. This is because synaptic current must first alter thecharge on the membrane capacitance of the muscle before themuscle membrane is depolarized (see Chapter 8 and Postscriptin this chapter).

ant cation tetramethylammonium (TMA) is around0.6 nm in diameter. In contrast, the voltage-gated Na +channel is only permeant to organic cations that aresmaller than 0.5 X 0.3 nm in cross section, and voltage-gated K+ channels will only conduct ions less than0.3 nm in diameter. The relatively large diameter of theACh pore is thought to provide a water-filled environ-ment that allows cations to diffuse through the channel,much as they would in free solution. This explains whythe pore does not discriminate between Na + and K+. It

also explains why even divalent cations, such as Ca2+,can permeate the channel. Anions are excluded, how-ever, by the presence of fixed negative charges in thechannel, as described later in this chapter.

The Current Flow Through Single IonChannels Can Be Measured by the Patch Clamp

The current for an end-plate potential flows throughseveral hundred thousand channels. Recordings of thecurrent flow through single ACh-gated ion channels, us-ing the patch clamp technique (see Box 6-1), have pro-vided us with insight into the molecular events under-lying the end-plate potential. Before the introduction ofthe patch clamp physiologists held two opposing viewsas to what the time course of the single-channel currentshould look like. Some thought that the single-channelcurrents were a microscopic version of the end-platecurrent recorded with the voltage clamp, having a rapid

Figure 11-7 The end-plate potential isproduced by the simultaneous flow ofNa+ and K+ through the same ACh-gated channels.A. The ionic currents responsible for theen~late potential can be determined bymeasuring the reversal potential of theen~late current. The voltage of the mus-cle membrane is clamped at different p0-tentials, and the synaptic current is mea-sured when the nerve is stimulated. If Na+flux alone were responsible for the end-plate current, the reversal potential wouldoccur at +55 mV, the equilibrium potentialfor Na+ (ENa!. The arrow next to each cur-rent record reflects the magnitude of thenet Na + flux at that membrane potential.

B. The end-plate current actually reversesat 0 mV because the ion channel is perme-able to both Na+ and K+, which are able tomove into and out of the cell simultane-ously (see Box 11-1). The net current is thesum of the Na+ and K+ fluxes through theend-plate channels. At the reversal poten-tial (EEPSP) the inward Na+ flux is balancedby an outward K+ flux so that no net cur-rent flows.

rising phase and a more slowly decaying falling phase.Others thought that the channels opened in an all-or-none manner, producing step-like currents similar tothat seen with gramicidin (see Chapter 6).

Individual Acetylcholine-Gated Channels Conducta Unitary Current

The first successful recordings of single ACh-gatedchannels from skeletal muscle cells, by Erwin Neherand Bert Sakmann in 1976, showed that the channelsopen and close in a step-like manner, generating verysmall rectangular steps of ionic current (Figure 11-8). Ata constant membrane potential a channel generates asimilar-size current pulse each time it opens. At a rest-ing potential of -90 m V the current steps are around-2.7 pA in amplitude. Although this is a very small cur-rent, it corresponds to a flow through an open channelof around 17 million ions per second!

The unitary current steps change in size with mem-brane potential. This is because the single-channel


A Hypothetical synapticcurrent due tomovement of Na+ only

B Synaptic currentreflecting movementof Na+ and K+

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current depends on the electrochemical driving force(V m - EEPSP)' Recall that Ohm's law applied to synaptic

current is

IHPSP = gEPSP X (V m - EHPSP)'

For single ion channels the equivalent expression is

iEPSP = 'YHPSP X (V m - EHPSP)'

where iEPSP is the amplitude of current flow throughone channel and 'YEPSP is the conductance of a singlechannel. The relationship between iEPSP and membranevoltage is linear, indicating that the single-channel con-ductance is constant and does not depend on mem-brane voltage; that is, the channel behaves as a simpleresistor. From the slope of this relation the channel isfound to have a conductance of 30 pS. The reversal p0-tential of 0 m V, obtained from the intercept of the volt-age axis, is identical to that for the end-plate current(Figure 11-9).

Although the amplitude of the current flowingthrough a single ACh channel is constant from opening


to opening, the duration of openings and the time be-tween openings of an individual channel vary consider-ably. These variations occur because channel openingsand closings are stochastic. They obey the same statisti-callaw that describes radioactive decay. Because of therandom thermal motions and fluctuations that a chan-nel experiences, it is impossible to predict exactly howlong it will take anyone channel to encounter ACh orhow long that channel will stay open before the AChdissociates and the channel closes. l:Iowever, the aver-age length of time a particular typf! of channel staysopen is a well-defined property of that channel, just asthe half-life of radioactive decay is an invariant prop-erty of a particular isotope. The mean open time forACh-gated channels is around 1 ms. Thus each channelopening is associated with the movement of about17,000 ions.

Unlike the voltage-gated channels, the ACh-gatedchannels are not opened by membrane depolarization.Instead a ligand (ACh) causes the channels to open. Eachchannel is thought to have two binding sites for ACh; toopen, a channel must bind two molecules of ACh. Once achannel closes, the ACh molecules dissociate and thechannel remains closed until it binds ACh again.

Four Factors Determine the End-Plate Current

Stimulation of a motor nerve releases a large quantity ofACh into the synaptic cleft. The ACh rapidly diffusesacross the cleft and binds to the ACh receptors, causingmore than 200,000 ACh receptor-channels to open al-most simultaneously. (This number is obtained by com-paring the total end-plate current, around - 500 nA,

with the current through a single ACh-gated channel,around -2.7 pA). How do small step-like changes incurrent flowing through 200,000 individual ACh-gatedchannels produce the smooth waveform of the end-plate current?

The rapid and large rise in ACh concentration uponstimulation of the motor nerve causes a large increase inthe total conductance of the end-plate membrane, gEPSP,and produces the rapid rise in end-plate current (Figure11-10). The ACh in the cleft falls to zero rapidly (in lessthan 1 IDs) because of enzymatic hydrolysis and diffu-sion. After the fall in ACh concentration, the channels be-gin to close in the random manner described above. Eachclosure produces a small step-like decrease in end-platecurrent because of the all-or-none nature of single-chan-nel currents. However, since each unitary current step is

Figure 11-8 Individual ACh-gatedchannels open in an all-or-none fash-ion.A. The patch~lamp technique is used torecord currents from single ACh-gatedchannels. The patch electrode is filledwith salt solution that contains a low con-centration of ACh and is then broughtinto close contact with the surface of themuscle membrane (see Box 6-1 ).B. Single-channel currents from a patchof membrane on a frog muscle fiberwere recorded in the presence of 100nM ACh at a resting membrane potentialof -90 mY. 1. The opening of a channelresults in the flow of inward current(recorded as a downward step). Thepatch contained a large number of ACh-gated channels so that successive open-ings in the record probably arise from dis-tinct channels. 2. A histogram of theamplitudes of these rectangular pulseshas a single peak. This distribution indi-cates that the patch of membrane con-tains only a single type of active channeland that the size of the elementary cur-rent through this channel varies randomlyaround a mean of -2.7 pA (1 pA =10-12 A). This mean, the elementarycurrent, is equivalent to an elementaryconductance of about 30 pS. (Courtesyof B. Sakmann.)C. When the membrane potential is in-creased to -130 mV, the individual chan-nel currents give rise to all-or-none incre-ments of -3.9 pA, equivalent to 30 pS.Sometimes more than one channelopens simultaneously. In this case, theindividual current pulses add linearly. Therecord shows one, two, or three chan- :,nels open at different times in responseto transmitter. (Courtesy of B. Sakmann.)

Chapter 11 / Signaling at the Nerve-Muscle Synapse: Directly Gated Thmsmission 195

A

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Acetytcholinereceptorchannels Cell membr8ne

B, Single-channel currents B2 Size of elementary current

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Figure 11-9 Single open ACh-gated channels behave assimple resistors.A. The voltage across a patch of membrane was systematicallyvaried during exposure to 2 fJ.M ACh. The current recorded atthe patch is inward at voltages negative to 0 mV and outwardat voltages positive to 0 mV, defining the reversal potential forthe channels.

tiny relative to the large current carried by many thou-sands of channels, the random closing of a large numberof small unitary currents causes the total end-plate cur-rent to appear to decay smoothly (Figure 11-10).

The summed conductance of all open channels in alarge population of ACh channels is the total synapticconductance, gEPSP = n X 'V, where n is the averagenumber of channels opened by the ACh transmitter and'V is the conductance of a single channel. For a largenumber of ACh channels, n = N X POI where N is the

total number of ACh channels in the end-plate mem-brane and Po is the probability that any given ACh chan-nel is open. The probability that a chaimel is open de-pends largely on the concentration of the transmitter atthe receptor, not on the value of the membrane poten-tial, because the channels are opened by the binding ofACh, not by voltage. The total end-plate current istherefore given by

IEPSI' = N x po x.., X (Vm - EEPSP)

or

IBPSJ> = n x.., X (Vm - EEPSP).

This equation shows that the current for the end-platepotential depends on four factors: (1) the total numberof end-plate channels (N); (2) the probability that a

B

B. The current flow through a single ACh-activated channel de-pends on membrane voltage. The linear relation shows that thechannel behaves as a simple resistor with a conductance ofabout 30 pS.

channel is open (Po); (3) the conductance of each openchannel ('Y); and (4) the driving force that acts on theions (V m - EEPSP)'

The relationships between single-channel current,total end-plate current, and end-plate potential areshown in Figure 11-11 for a wide range of membrane

potentials.

The Molecular Properties of the Acetylcholine-Gated Channel at the Nerve-Muscle SynapseAre Known

Ligand-Gated Channels for Acetylcholine DifferFrom Voltage-Gated Channels

Ligand-gated channels such as the ACh-gated channelsthat produce the end-plate potential differ in two im-portant ways from the voltage-gated channels that gen-erate the action potential at the neuromuscular junction.First, two distinct ~ of voltage-gated channels areactivated sequentially to generate the action potential,one selective for Na + and the other for K+. In contrast,

the ACh-gated channel alone generates end-plate p0-tentials, and it allows both Na + and K+ to pass with

nearly equal permeability.

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A second difference between ACh-gated and volt-age-gated channels is that Na + flux through voltage-

gated channels is regenerative: the increased depolar-ization of the cell caused by the Na + influx opens morevoltage-gated Na + channels. This regenerative feature

is responsible for the all-or-none property of the actionpotential. In contrast, the number of ACh-activatedchannels opened during the synaptic potential variesaccording to the amount of ACh available. The depolar-ization produced by Na + influx through these channels

does not lead to the opening of more transmitter-gatedchannels; it is therefore limited and by itself cannot pro-duce an action potential. To trigger an action potential, asynaptic potential must recruit neighboring voltage-gated channels (Figure 11-12).

As might be expected from these two differences inphysiological properties, the ACh-gated and voltage-gated channels are formed by distinct macromoleculesthat exhibit different sensitivities to drugs and toxins.Tetrodotoxin, which blocks the voltage-gated Na + chan-nel, does not block the influx of Na + through the nico-

tinic ACh-gated channels. Similarly, a-bungarotoxin, asnake venom protein that binds tightly to the nicotinicreceptors and blocks the action of ACh, does notinterfere with voltage-gated Na + or K+ channels

(a-bungarotoxin has proved useful in the biochemicalcharacterization of the ACh receptor).

In Chapter 12 we shallieam about still another typeof ligand-gated channel, the N-methyl-D-aspartate orNMDA-type glutamate receptor, which is found in mostneurons of the brain. This channel is doubly gated, re-sponding both to voltage and to a chemical transmitter.

A Single Macromolecule Forms the NicotinicAcetylcholine Receptor and Channel

The nicotinic ACh-gated channel at the nerve-musclesynapse is a directly gated or ionotropic channel: thepore in the membrane through which ions flow and thebinding site for the chemical transrfrltter (ACh) that reg-ulates the opening of the pore are all formed by a singlemacromolecule. Where in the molecule is the bindingsite located? How is the pore formed? What are its prop-erties? Insights into these questions have been obtainedfrom molecular studies of the ACh-gated receptor-chan-nel proteins and their genes.

Biochemical studies lw Arthur Karlin and Jean-Pierre Changeux indicate,r'hat the mature nicotinic AChreceptor is a membrane glycoprotein formed from fivesubunits: two a-subunits and one 13-, one 'V-, and one 8-subunit (Figure 11-13). The amino terminus of each ofthe subunits is exposed on the extracellular surface ofthe membrane. The amino terminus of the a-subunit


A Idealized time course of opening of six ion channels

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B Total current of the six channels

T3~I 2ma

FIgure 11-10 The time course of the total current at theend-plate results from the summed contributions of manyindividual ACh-gated channels. (Adapted from D. Colquhoun1981.)A. Individual ACh-gated channels open in response to a briefpulse of ACh. All channels (1-6) open rapidly and nearly simulta-neously. The channels then remain open for varying durationsand close at different times.B. The stepped trace shows the sum of the six records in A. Itreflects the sequential closing of each channel (the number in-dicates which channel has closed) at a hypothetical end-platecontaining only six channels. In the final period of net currentflow only channel 1 is open. In a current record from a wholemuscle fiber, with thousands of channels, the individual chan-nel closings are not visible because the total end-plate current(hundreds of nanoamperes) is so much larger than the single-channel current amplitude (-2.7 pAl. As a result, the total end-plate current appears to decay smoothly.

contains a site that binds ACh with high affinity. Karlinand his colleagues have demonstrated the presence oftwo extracellular binding sites for ACh on each channel.Those sites are formed in a cleft between each a-subunitand its neighboring 1- or &-subunits. One molecule ofACh must bind to each of the two a-subunits for thechannel to open efficiently (Figure 11-13). The inhibitorysnake venom a-bungarotoxin also binds to the a-subunit.

198 Part ill / ElementaryInteractions

A End-plate Total end1)18te Slngl8-channel B ACh-geted Restingpotential current current ch8MeI. ch8I'InIIt

+30mV ~

EEJ'SP 0 mY

Restingmembranepotential

Figure 11-11 Membrane potential affects the end-plate po-tential, total end-plate current, and ACh-gated single-chan-nel current in a similar way.A. At the normal muscle resting potential of -90 mV the sin-.gle-channel currents and total en~late current (made up ofcurrents from more than 200,0000 channels) are large and in-ward because of the large inward driving force on current flowthrough the ACh1Jated channels. This large inward current pro-duces a large depolarizing end-plate potential. At more positivelevels of membrane potential (increased depol~rization), the in-ward driving force on Na + is less and the outwdrd driving force

on K+ is greater. This results in a decrease in the size of thesingle-channel currents and in the magnitude of the en~latecurrents, thus reducing the size of the en~late potential. Atthe reversal potential (0 mV) the inward Na+ flux is balanced bythe outward K+ flux, so there is no net current flow at the end-

Insight into the structure of the channel pore hascome from analysis of the primary amino add se-quences of the receptor-channel su1]units as well asfrom biophysical studies. The workpf Shosaku Numaand his colleagues demonstrated that the four subunit

Between Neurons: Synaptic Transmission

-. 1'0-

J

t

t

t

t, 0,-

~~

~ J1.JL..JL

~ t

~ t ~

.

plate and no change in Vm. Further depolarization to +30 mVinverts the direction of the end-plate current, as there is now alarge outward driving force on K+ and a small inward drivingforce on Na+. As a result, the outward flow of K+ hyperpolar-izes the membrane. On either side of the reversal potential theend-plate current drives the membrane potential toward the re-versal potential.

B. The direction of Na+ and K+ fluxes in individual channels isaltered by changing Vm. The algebraic sum of the Na+ and K+currents, IN. and IK, gives the net CUffent that flows throughthe ACh1;Jated channels. This net synaptic current is equal insize, and opposite in direction, to that of the net extrasynapticcurrent flowing in the return pathway of the resting channelsand membrane capacitance. (The length of each arrow repre-sents the relative magnitude of a current)

types are encoded by distinct but related genes. Se-quence comparison of the subunits shows a high degreeof similarity among them: half of the amino acidresidues are identical or conservatively substituted.This similarity suggests that all subunits have a similar

structure. Furthermore, all four of the genes for the sub-units are homologous; that is, they are derived from acommon ancestral gene.

The distribution of the polar and nonpolar aminoacids of the subunits provides important clues as to howthe subunits are threaded through the membrane bi-layer. Each subunit contains four hydrophobic regionsof about 20 amino acids called M1-M4, each of which isthought to form an a-helix traversing the membrane.The amino acid sequences of the subunits suggest thatthe subunits are symmetrically arranged to create thepore through the membrane (Figure 11-14).

The walls of the channel pore are thought to beformed by the M2 region and by the segment connect-ing M2 to M3 (Figure 11-148). Certain drugs that bind toone ring of serine residues and two rings of hydropho-bic residues on the M2 region within the channel poreare able to inhibit current flow through the pore. More-over, three rings of negative charge that flank the M2 re-gion (Figure 11-158) contribute to the channel's selectiv-ity for cations. Each ring is made up of three or fouraligned negatively charged residues contributed by dif-ferent subunits.

A three-dimensional model of the ACh receptor hasbeen proposed by Arthur Karlin and Nigel Unwinbased on neutron scattering and electron diffraction im-ages respectively (see Figure 11-3). The receptor-channelcomplex is divided into three regions: a large vestibuleat the external membrane surface, a narrow transmem-brane pore that determines cation selectivity, and alarger exit region at the internal membrane surface (Fig-ure 11-15A). The region that extends into the extracellu-lar space is surprisingly large, about 6 nm in length. Atthe external surface of the membrane the channel has awide mouth about 2.5 nm in diameter. Within the bi-layer of the membrane the channel gradually narrows.

This narrow region is quite short, only about 3 nm inlength, corresponding to the length of both the M2 seg-ment and the hydrophobic core of tJ;1e bilayer (Figure 11-158). In the open channel, the M2 "segment appears toslope inward toward the central axis of the channel, so thatthe pore narrows continuously from the outside of themembrane to the inside (Figure 11-1SC). Near the innersurface of the membrane the pore reaches its narrowest di-ameter, around 0.8 nm, in reasonable agreement with esti-mates from electrophysiological measurements. This sitemay correspond to the selectivity filter of the channel. Atthe selectivity filter, polar threonine residues extend theirside chains into the lumen of the pore. The electronegativeoxygen atom of the hydroxyl group may interact with thepermeant cation to compensate for loss of waters of hy-dration. At the inner surface of the membrane, the poresuddenly widens again.


,.,

ACh binding attnlnsmitter-getedchennela

+ChIonel opening

+Ne+ inflowK+ outflow

+DepoIerIZItionl(end1:II8te potential)

Opening of~Ne+ rnnelS

)Na + inflow

+Depolarization

+Action potential

AOI

ACh-liltedcllenr

Figure 11.12 The binding of ACh in a postsynaptic musclecell opens channels permeable to both Na+ and K+. Theflow of these ions into and out of the cell depolarizes the cellmembrane, producing the end-plate potential. This depolariza-tion opens neighboring voltage-gated Na + channels in the mus-

cle cell. To trigger an action potential, the depolarization pro-duced by the end-plate potential must open a sufficient numberof Na+ channels to exceed the cell's threshold. (After Alberts etal. 1989.)

/

200 Part m / Elementary Interactions Between Neurons: Synaptic Transmission

figure 11-13 Three-dimensional model of the nicotinicACh-gated ion channel. The receptor-channel complex con-sists of five subunits. all of which contribute to forming thepore. lNhen two molecules of ACh bind to portions of the a-subunits exposed to the membrane surface. the receptor-

On the basis of images of ACh receptors in the pres-ence and absence of transmitter, Unwin has proposedthat the M2 helix may be important for channel gating,as well as for ion permeation. His studies indicate thatthe M2 helix is not straight but rather has a bend or kinkin its middle (Figure 11-15C). When the channel isclosed, this kink projects inward toward the central axisof the pore, thereby occluding it. When the channelopens, the M2 helix rotates so that the kink lies along thewall of the channel.

A somewhat different view of the pore and gate hasbeen provided by Karlin, who studied the reactions ofsmall, charged reagents with amino aCid side chains inthe M2 segment. By comparing the ability of these com-pounds to react in the open and closed states of the

Agu,. 11-14 A molecular model of the transmembranesubunits of the nicotinic ACh receptor-channel.

A. Each subunit is composed of four membrane-spanninga-helices (labeled M1 through M4).B. The five subunits are arranged such that they form an aque-ous channel, with the M2 segment of each subunit facing in-side end forming the lining of the pore (see turquoise cylinders,Figure 1 1-15A). Note that the ..,..subunit lies between the two ex-subunits.

Two N:1II'11C111c:1M8 bound:ChInneI open

No ACh bound:Ch8nn8I cIoI8d

.NIl>

channel changes conformation. This opens a pore in the portionof the channel embedded in the lipid bilayer. and both K+ andNa + flow through the open channel down their electrochemical

gradients.

A A single subunit in the ACh receptor-(:hannel

~!;!2

~OOH

CytopIIImic Iide

\B Hypothetical arrangement of subunits in

one channel

ExtrK8IIuI8rside

CytOplllsmic:side

A Functional model of AChreceptor-channel

ExtT8celiulerside

C Structural model for channel gating

Closed

Figure 11-15 A functional model ofthe nicotinic AChreceptor-channel.A. According to this model negatively charged amino acids oneach subunit form three rings of charge around the pore (seepart B). As an ion traverses the channel it encounters this se-ries of negatively charged rings. The rings at the external (1)and internal (3) surfaces of the cell membrane may serve asprefilters and divalent blocking sites. The central ring (2) withinthe bilayer may contribute to the selectivity filter for cations.along with a ring of threonine and serine residues that con-tribute an electronegative oxygen. (Dit;nensions are not toscale.)B. The amino acid sequences of the M2 and flanking regions ofeach of the five subunits. The horizontal series of amino acids

channel, Karlin concluded that the gate was at the cyto-plasmic end of M2.

An Overall View

The terminals of mptdf neurons form synapses withmuscle fibers at specialized regions in the muscle mem-


B Amino acid sequence of channelsubunits

Subunit a ya 6 p.I I Ie .I .Ilie VIi /1" ' l,ey 'M

~IIII~:2 ,~~",

I~!V I L I

'Gly Gly oGlyGIy QIy: Cytoplasmic

3 ;;;.;Sid8thr~. Thr AlII ~'I 1 I I 1

Funnel shapedentrance region

Exit region Ion selective pofe

o,.n

numbered 1, 2, and 3 identify the three rings of negativecharge (see part A). The position of the aligned serine and thre-onine residues within M2, which help form the selectivity filter,is indicated.C. A model for gating of the ACh receptor-channel. Three of thefive M2 transmembrane segments are shown. Each M2 seg-ment is split into two cylinders, one on top of the other. Left: Inthe closed state each M2 cylinder points inward toward thecentral axis of the channel. A ring of five hydrophobic leucineresidues (large spheres, one from each M2 segment) occludesthe pore. Right In the open state the cylinders tilt, thus enlarg-ing the ring of leucines. A ring of hydrophilic threonine residues(small spheres) may form the selectivity filter near the innermouth of the channel. (Based on Unwin 1995).

brane called end-plates. When an action potentialreaches the terminals of a presynaptic motor neuron, itcauses release of ACh. The transmitter diffuses acrossthe synaptic cleft and binds to nicotinic ACh receptorsin the end-plate, thus oPening channels that allow Na +,K+, and ea2+ to flow across the postsynaptic muscle. Anet influx of Na + ions produces a depolarizing synaptic

potential called the end-plate potential.


Because the ACh-activated channels are localized tothe end-plate, the opening of these channels producesonly a local depolarization that spreads passively alongthe muscle fibers. But by depolarizing the postsynapticcell past threshold, the transmitter-gated channels acti-vate voltage-dependent Na + channels near the end-

plate region. As the postsynaptic cell becomes progres-sively depolarized, more and more voltage-gated Na +channels open. In this way the Na + channels can

quickly generate enough current to produce an activelypropagated action potential.

The protein that forms the nicotinic ACh-activatedchannel has been purified, its genes cloned, and itsamino acids sequenced. It is composed of five subunits,two of which-the a-subunits that recognize and bindACh-are identical. Each subunit has four hydrophobicregions that are thought to form membrane-spanninga-helices.

The protein that forms the nicotinic ACh-gatedchannel also contains a site for recognizing and bind-ing the ACh. This channel is thus gated directly by achemical transmitter. The functional molecular do-mains of the ACh-gated channel have been identified,and the steps that link ACh-binding to the opening ofthe channel are now being investigated. Thus, wemay soon be able to see in atomic detail the molecu-lar dynamics of this channel's various physiologicalfunctions.

The large number of ACh-gated channels at theend-plate normally ensures that synaptic transmissionwill proceed with a high safety factor. In the autoim-mune disease myasthenia gravis, antibodies to the AChreceptor decrease the number of ACh-gated channels,thus seriously compromising transmission at the neuro-muscular junction (see Chapter 16).

Acetylcholine is only one of many neurotransmit-ters in the nervous system, and the end-plate potentialis just one example of chemical signaling. Do trans-mitters in the central nervous system. act in the samefashion, or are other mechanisms involved? In thepast such questions were virtually unanswerable be-cause of the small size and great variety of nerve cellsin the central nervous system. However, advances in

experimental technique-in particular, patch clamp-ing-have made synaptic transmission at centralsynapses easier to study. Already it is clear that manyneurotransmitters operate in the central nervous sys-tem much as ACh operates at the end-plate, whileother transmitters produce their effects in quite differ-ent ways. In the next two chapters we shall exploresome of the many variations of synaptic transmissionthat characterize the central and peripheral nervous

systems.

Postscript: The End-Plate Current Can BeCalculated From an Equivalent Circuit

Although the flow of current through a population ofACh-activated end-plate channels can be described byOhm's law, to understand fully how the flow of electri-cal current generates the end-plate potential we alsoneed to consider all the resting channels in the sur-rounding membrane. Since channels are proteins thatspan the bilayer of the membrane, we must also takeinto consideration the capacitive properties of the mem-brane and the ionic batteries determined by the distrib-ution of Na + and K+ inside and outside the cell.

The dynamic relationship of these various compo-nents can be explained using the same rules we used inChapter 8 to analyze the flow of current in passive elec-trical devices that consist only of resistors, capacitors,and batteries. We can represent the end-plate regionwith an equivalent circuit that has three parallelbranches: (1) a branch representing the flow of synapticcurrent through the transmitter-gated channels; (2) abranch representing the return current flow throughresting channels (the nonsynaptic membrane); and (3) abranch representing current flow across the lipid bi-layer, which acts as a capacitor (Figure 11-16).Since the end-plate current is carried by both Na +

and K+, we could represent the synaptic branch of theequivalent circuit as two parallel branches, each repre-senting the flow of a different ion species. At the end-plate, however, Na + and K+ flow through the same ion

channel. It is therefore more convenient (and correct) tocombine the Na + and K+ current pathways into a single

conductance, representing the channel gated by ACh.The conductance of this pathway depends on the num-ber of channels opened, which in turn depends on theconcentration of transmitter. In the absence of transmit-ter no channels are open and the conductance is zero.When a presynaptic action potential causes the releaseof transmitter, the conductance of this pathway rises to avalue of around 5 X 10-6 S (or a resistance of 2 x 1<f m.This is about five times the conductance of the parallelbranch representing the resting or leakage channels (gl)'

The end-plate conductance is in series with a bat-tery (EEPSP)' whose value is given by the reversal poten-tial for synaptic current flow (0 mY) (Figure 11-16). Thisvalue is the weighted algebraic sum of the Na + and K+

equilibrium potentials (see Box 11-1).The current flowing during the excitatory post-

synaptic potential (IEPSP) is given byIEPSP = gBPSP X (V m - EEPSP)'

Using this equation and the equivalent circuit of Figure11-17 we can now analyze the end-plate potential in

Figure 11-18 The equivalent circuit of the end-plate withtwo parallel current pathways. One pathway representingthe synapse consists of a battery, EEPsP, in series with a con-ductance through ACh-gated channels, Q'epsP. The other path-way consists of the battery representing the resting potential(EI) in series with the conductance of the resting channels (g).In parallel with both of these conductance pathways is themembrane capacitance (c'",). The voltmeter M measures thepotential difference between the inside and the outside of thecell.

When no ACh is present, the gated channels are closed andno current flows through them. This state is depicted as anopen electrical circuit in which the synaptic conductance is notconnected to the rest of the circuit. The binding of ACh opensthe synaptic channel. This event is electrically equivalent tothrowing the switch that connects the gated conductance path-way (YEpsp) with the resting pathway (Q). In the steady statecurrent flows inward through the gated channels and outwardthrough the resting channels. With the indicated values of con-ductances and batteries, the membrane will depolarize from-90 mV (its resting potential) to -15 mV (the peak of the end-plate potential).

terms of the flow of ionic current. At the onset of the ex-citatory synaptic action (the dynamic phase), an inwardcurrent (IEPSP) flows through the ACh-activated chan-nels because of the increased coriductance to Na + andK+ and the large inward driving force on Na + at the ini-

tial resting potential (-90 mY). Since current flows in aclosed loop, the inward synaptic current must leave thecell as outward current. From the equivalent circuit wesee that there are two parallel pathways for outwardcurrent flow: a conductance pathway (II) representingcurrent flow through the resting (or leakage) channelsand a capacitive pathway (Ie) representing current flowacross the membrane capacitance. Thus,

IBPSP = -UI + lJ.

During the earliest phase of the end-plate potentialthe membrane poten~l, V D\I is still close to its resting

203Chapter 11 / Signaling at the Nerve-Muscle Synapse: Directly Gated Transmission

value, EI' As a result, the outward driving force on cur-rent flow through the resting channels (V m - EI) issmall. Therefore, most of the current leaves the cell ascapacitive current and the membrane depo1arizesrapidly (phase 2 in Figure 11-17). As the cell depo1arizes,the outward driving force on current flow through theresting channels increases, while the inward drivingforce on synaptic current flow through the ACh-gatedchannels decreases. Concomitantly, as the concentrationof ACh in the synapse falls, the ACh-gated channels be-gin to close, and eventually the flow of inward currentthrough the gated channels is exactly balanced by out-ward current flow through the resting channels UEPSP =- II)' At this point no current flows into or out of the ca-pacitor, that is, Ic = O. Since the rate of change of mem-brane potential is directly proportional to Ic,

Ic = Cm x ~V/~t,

the membrane potential will have reached a peaksteady-state value, ~V / ~t = 0 (phase 3 in Figure 11-17).

As the gated channels close, IEPSP decreases further.Now IEPSp and II are no longer in balance and the mem-brane potential starts to repolarize, because the outwardcurrent flow due to II becomes larger than the inwardsynaptic current. During most of the declining phase ofthe synaptic action, current no longer flows through theACh-gated channels since all these channels are closed.Instead, current flows out only through the restingchannels and in across the capacitor (phase 4 in Figure11-17).

When the end-plate potential is at its peak orsteady-state value, Ic = 0 and therefore the value of V mcan be easily calculated. The inward current flowthrough the gated channels (1EPSP) must be exactly bal-anced by outward current flow through the restingchannels (11):

IEPSP + II = O. (11-8)

The current flowing through the active ACh-gated chan-nels (IEPSP) and through the resting channels (II) is givenbyahm'slaw:

IEPSp = gEPSP X (V m - EEPSP)'

and

II = gl X (V m - EI).

By substituting these two expressions into Equation11-8, we obtain

gEPSP X (Vm - EEPSP) + gl X (Vm - EI) = O.

To solve for V m we need only expand the two productsin the equation and rearrange them so that all terms in

Part ill / Elementary Interactions Between Neurons: Synaptic Transmission204

A

End-pl1t8potential

Current

B Time 1Steady state

'.-0_-0

Figure 11-17 Both the ACh-gated synaptic conductance andthe passive membrane properties of the muscle cell deter-mine the time course of the end-plate potential.A. Comparison of the time course of the end-plate potential(top trace) with the time courses of the component currentsthrough the ACh-gated channels (/EPSp). the resting (or leakage)channels W. and the capacitor (/J. Capacitive current flowsonly when the membrane potential is changing. In the steady

Time'

Dynamic(decline of synaptic action)

Time 3Steady state

(peak of synaptic action)

Time 2Dynemic

(onset of synaptic actionl

."state, such as at the peak of the end-plate potential, the inwardflow of ionic current through the ACh-gated channels is exactlybalanced by the outward flow of ionic current across the rest-ing channels, and there is no flow of capacitive current.B. Equivalent circuits for the current at times 1, 2, 3, and 4shown in part A. (The relative magnitude of a current is repre-sented by the length of the arrows.)

voltage (V m) appear on the left side:

(gBPSP x V m) + (gl x V m) = (gBPSP x EBPSP) + (gl x E1).

By factoring out V m on the left side, we finally obtain

(gEPSP x EEPSP) + (gl x E1)V m = + . (11-9)

gBPSP gl

This equation is similar to that used to calculate theresting and action potentials (Chapter 7). According toEquation 11-9, the peak voltage of the end-plate poten-tial is a weighted average of the electromotive forces ofthe two batteries for gated and resting currents. Theweighting factors are given by the relative magnitude ofthe two conductances. If the gated conductance is muchsmaller than the resting membrane conductance(gEPSP <c gl), gEPSP X EEPSP will be negligible comparedwith gl X EI' Under these conditions, V m will remainclose to EI' This situation occurs when only a few chan-nels are opened by ACh (because its concentration islow). On the other hand, if gEPSP is much larger than gl1Equation 11-9 states that V m approaches EEPSP, the

synaptic reversal potential. This situation occurs whenthe concentration of ACh is high and a large number ofACh-activated channels are open. At intermediate AChconcentrations, with a moderate number of ACh-activated channels open, the peak end-plate potentiallies somewhere between EI and EEPSP'

We can now use this equation to calculate the peakend-plate potential for the specific case shown in Figure11-16, where gEPSP = 5 X 10-6 S, gl = 1 X 10-6 S, EEPSP =0 m V, and EI = -90 m V. Substituting these values into

Equation 11-9 yields

VIII =[(5 X 10-6 S) X (0 mV)} + [(1 X 10-6 S) X (-90 mV)]

(5 x 10-6S) + (1 X 10-6S) .

or

(1 X 10-6 S) X t-90 mY)Vm = (6 X 10 6S)

= -15 mY.

The peak amplitude of the end-plate potential is then

.:1VEPSP = Vm - El

= -15 mV - (-90 mY)

-75mV.

As a check for consistency we can see whether, atthe peak of the end-plate potential, the synaptic currentis equal and opposite to the nonsynaptic current so thatthe net membrane current is indeed equal to zero:

"

Chapter 11 / Signaling at the Nerve-Muscle Synapse: Directly Gated li'ansmission 205

IEPSP = (5 x 10-65) X (-15mV - OmV)

= -75 X 10-9 A

I) = (1 X 10-65) X [-15mV - (-90mV)],

= 75 X 10-9 A.

we see that Equation 11-9 ensures

and

Here thatIEPSP + II = O.

Eric R. KandelSteven A. Siegelbaum

Selected Readings

Fatt P, Katz B. 1951. An analysis of the end-plate potentialrecorded with an intra-ce1lular electrode. J Physiol (Land)115:320-370.

Heuser JE, Reese 1'5.1977. Structure of the synapse. In: ERKandel (ed), Handbook of PhysiohJgy: A Critical, Comprehen-sive Presentation of Physiological Knowledge and Concepts,Sect. 1, The Nervous System. Vol. 1, Cellular Biology of Neu-rons, Part 1, pp. 261-294. Bethesda, MD: American Physi-ological Society. .

Hille B. 1992. In: Ionic Channels of Excitable Membranes, 2nded, pp. 140-169. Sunderland, MA: Sinauer.

Imoto K, Busch C, Sakmann B, Mishina M, Konno T, Nakai J,Bujo H, Mori Y, Fukuda K, Numa S. 1988. Rings of nega-tively charged amino acids determine the acetylcholinereceptor-channel conductance. Nature 335:645-648.

Karlin A, Abbas MH. 1995. Toward a structural basis for thefunction of nicotinic acetylcholine receptors and theircousins. Neuron 15:1231-1244.

Neher E, Sakmann B. 1976. Single-channel currents recordedfrom membrane of denervated frog muscle fibres. Nature260:799-802.

Unwin N. 1993. Neurotransmitter action: opening of ligand-gated ion channels. Cell 72(Suppl):31-!1.

References

Abbas MH, Kaufmann C, Archdeacon P, Karlin A. 1994.Identification of acetylcholine receptor-channel-liningresidues in the entire M2 segment of the «-subunit. Neu-ron 13:919-927.

Alberts B, Bray D, Lewis J, Raft M, Roberts K, Watson JD.1989. Molecular Biology of the Cell, 2nd ed. New York:Gubmd. .

Charnet P, Labarca C, Leonud RJ, Vogelau NJ, Czyzyk L,Gouin A, Davidson N, Lester HA. 1990. An open clwmel

206 Interactions Between Neurons: Synaptic TransmissionPart ill / Elementary

blocker interacts with adjacent turns of a-helices in thenicotinic acetylcholine receptor. Neuron 4:87-95.

Claudio T, Ballivet M, Patrick J, Heinemann S. 1983.Nucleotide and deduced amino acid sequences of Torpedocalifornica acetylcholine receptor 'Y-subunit. Proc NatlAcad Sci U SA80:1111-1115.

Colquhoun O. 1981. How fast do drugs work? 'fiends Phar-macol Sci 2:212-217.

Dwyer TM, Adams OJ, Hille B. 1980. The permeability of theendplate channel to organic cations in frog muscle. J GenPhysiol 75:469-492.

Fertuck HC, Salpeter MM. 1974. Localization of acetyl-choline receptor by l~-labeled a-bungarotoxin bindingat mouse motor endplates. Proc Natl Acad Sci USA71:1376-1378.

Heuser IE, Salpeter SR. 1979. Organization of acetylcholinereceptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell BioI82:150-173.

Ko C-P. 1984. Regeneration of the active zone at the frog neu-romuscular junction. J Cell Bioi 98:1685-1695.

Kuffler SW, Nicholls JG, Martin AR. 1984. From Neuron toBrain: A Cellular Approach to the Function of the Nervous Sys-tem, 2nd ed. Sunderland, MA: Sinauer.

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Shimizu S, Kikyotani S, Kayano T, Hirose T, Inayama S,Numa S. 1983. Cloning and sequence analysis of calfcONA and human genomic DNA encoding a-subunitprecursor of muscle acetylcholine receptor. Nature305:818-823.

Noda M, Takahashi H, Tanabe T, Toyosato M, Kikyotani 5,Furutani Y, Hirose T, Takashima H, Inayama 5, Miyata T,Numa S. 1983. Structural homology of Torpedo californicaacetylcholine receptor subunits. Nature 302:528-532.

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Raftery MA, Hunkapiller MW, Strader CD, Hood LB. 1980.Acetylcholine receptor. complex of homologous subunits.Science 208:1454-1457.

Revah F, Galzi J-L, Giraudat J, Haumont FY, Lederer F,Changeux J-P. 1990. The noncompetitive blocker[~chlorpromazine labels three amino acids of theacetylcholine receptor gamma subunit: implications forthe alpha-helical organization of regions Mll and for thestructure of the ion channel. Proc Natl Acad Sci USA

87:4675-4679.Takeuchi A. 1977. Junctional transmission. I. Postsynaptic

mechanisms. In: ER Kandel (ed), Handbook of Physiology: ACritical, Comprehensive Presentation of Physiological Knowl-edge and Concepts, Sect. 1, The Nervous System. Vol. 1, Cellu-lar Biology of Neurons, Part 1, pp. 295-327. Bethesda, MD:American Physiological Society.

Toyoshima C, Unwin N. 1988. Ion channel of acetylcholinereceptor reconstructed from images of postsynaptic mem-branes. Nature 336:247-250.

Verrall 5, Hall ZW. 1992. The N-terminal domains of acetyl-choline receptor subunits contain recognition signals forthe initial steps of receptor assembly. Cell 68:23-31.

Vtllarroel A, Herlitze 5, Koenen M, Sakmann B. 1991. Loca-tion of a threonine residue in the alpha-subunit M2 trans-membrane segment that determines the ion flow throughthe acetylcholine receptor-channel. Proc R Soc Lond BBioI Sci 243:69-74.

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'"

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