Proc. Nati. Acad. Sci. USAVol. 77, No. 11, pp. 6912-6916, November 1980Neurobiology

Mechanism of calcium current modulation underlying presynapticfacilitation and behavioral sensitization in Aplysia

(learning/synaptic plasticity/serotonin/cyclic AMP/potassium currents)

MARC KLEIN AND ERIC R. KANDELDepartments of Physiology and Psychiatry, Division of Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York,New York 10032; and The New York State Psychiatric Institute, 722 West 168th Street, New York, New York 10032

Contributed by Eric R. Kandel, August 20,1980

ABSIRACT Behavioral sensitization of the gill-withdrawalreflex of Aplysia is caused by presynaptic facilitation at thesynapses of the mechanoreceptor sensory neurons of the reflexonto the motor neurons and interneurons. The presynaptic fa-cilitation has been shown to be simulated by serotonin (theputative presynaptic facilitatory transmitter) and by cyclic AMPand to be accompanied by an increase in the Ca2+ current ofsensory neuron cell bodies exposed to tetraethylammonium.This increase in the Ca2+ current could result from either a di-rect action on the Ca2+ channel or an action on an opposing K+current. Here we report voltage clamp experiments which in-dicate that the increase in Ca2+ current associated with presy-naptic facilitation results from a decrease in a K+ current.Stimulation of the connective (the pathway that mediates sen-sitization) or application of serotonin causes a decrease in avoltage-sensitive, steady-state outward current measured undervoltage clamp as well as an increase in the transientnetinwardand a decrease in the transient outward currents elicited by briefdepolarizing command steps. The reversal potential of thesteady-state synaptic current is sensitive to extracellular K+concentration, and both the steady-state synaptic current andthe changes in the transient currents are blocked by K+ currentblocking agents and by washout of KV. These results suggest thatserotonin and the natural transmitter released by connectivestimulation act to decrease a voltage-sensitive K+ current. Thedecrease in K+ current prolongs the action potential, and thisin turn increases the duration of the inward Ca2+ current andthereby enhances transmitter release.

Behavioral sensitization, a simple form of learning, is an increasein reflex responsiveness that follows the presentation of a novelstimulus. Sensitization of the gill-withdrawal reflex of Aplysiais caused by presynaptic facilitation of synaptic transmissionbetween the sensory neurons and the motor and interneuronsof the reflex pathway (1). Brunelli et al. (2) found that thepresumptive facilitatory neurotransmitter is serotonin or a re-lated substance (see also refs. 14 and 15) and that it producespresynaptic facilitation by increasing the concentration of cyclicAMP (cAMP) in the sensory neurons (2-4).We have presented evidence (5) that presynaptic facilitation

at these synapses is associated with an increase in the Ca2+component of the action potentials of the sensory neuron andthat the increase in the presynaptic Ca2+ current could also beproduced by serotonin, cAMP, or phosphodiesterase inhibitors.We therefore proposed that the increase in transmitter releaseunderlying sensitization is caused by an increase in Ca2+ influxinto the terminals that is mediated by cAMP.

These earlier experiments did not allow us to specify whetherthe increase in the Ca2+ current was the result of a direct actionof the facilitatory transmitter on the Ca2+ channels of thepresynaptic terminals or was caused by an increase in duration

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

6912

of the sensory neuron action potential resulting from a synap-tically mediated decrease in K+ current. The results of the ex-periments described below indicate that presynaptic facilitationis caused by prolongation of the presynaptic action potentialresulting from a decrease in K+ current.

METHODSAplysia californica weighing 70-250 g were used. For voltageclamping, sensory cells were impaled with two independentelectrodes (resistance, 5-20 MU) and a Dagan 8500 voltage-clamp was used. When the potential was stepped to a new level,the membrane voltage settled to its final value within less than700 p-sec. In some of the voltage-clamp experiments, cell bodies

--wereisolated from their processes by ligation.Solutions and Drugs. The "normal seawater" had the fol-

lowing composition (mM): NaCl, 460; KCl, 10; CaC12, 11;MgCI2, 55; Tris buffer (TRIZMA, pH 7.6; Sigma), 10. Highdivalent cation solution contained (mM): NaCl, 265; KCl, 10;CaC12, 60; MgCl2, 140; and Tris, 10. Other experimental solu-tions contained tetraethylammonium chloride (Et4NCl, East-man), 4-aminopyridine (Sigma), or barium, nickel, or cobaltions, as indicated. At least 10 bath volumes of each experimentalsolution was perfused through the chamber before an experi-ment was begun. Other drugs used in these studies includednystatin (Sigma) and serotonin creatinine sulfate (ICN).

Nystatin was dissolved to 5 mg/ml in methanol and thendiluted to 40 mg/liter in 300 mM CsCl/100 mM MgSO4/400mM sucrose/10 mM Tris. Ganglia were bathed in this solutionfor 20-30 min. Ganglia were bathed for an additional 40-45min in an identical solution but without nystatin for washout.Preparations were then superfused with an experimental so-lution containing 11 mM CaCl2, 55 mM MgCl2, and 480 mMTris (6, 7).

RESULTSPresynaptic Facilitation Is Associated with a Decrease

in Steady-State K+ Conductance. When a sensory neuron isvoltage clamped in normal seawater and the connective (thepathway from the head that mediates sensitization) is stimu-lated, the holding current moves inward for several minutes(Fig. 1A1). This shift of the holding current corresponds to thelong-lasting depolarizing synaptic potential observed in anunclamped cell (5). In this study we did not examine the timecourse of this current beyond 10 min, but we have reason tobelieve that it can last more than 30 min (5). To examine theresting membrane conductance we imposed small hyperpo-larizing command steps and found that connective stimulationand serotonin caused a decrease in the elicited inward current,

Abbreviations: cAMP, cyclic AMP; Et4N+, tetraethylammonium;EPSP, excitatory postsynaptic potential.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 7,

202

0

Proc. Natl. Acad. Sci. USA 77 (1980) 6913

Al CS

12.5 nA

a b

Vm1J2 mV10 sec

A2Im ~~~~~~~~_--------

a fib J12.5 nAVm 100 msec-50 mV-70 -j LF

3-

2

1 VM, MV ,'-80 -70-60 / o

0o ,,~I ,-0 - - 40 -3c-1 --

C._

C,

-I

04

CQ

w

-1

-2

-3.

C1 Vm, mV

-80 -70 -60 -50 -40 -30a. -iII ~-'ox

\a

4- B2

3]2]

1t V, mV ,/1-80-70-60 -

n .

-1 -

25-.¢ 20-r 15-

j 10-5-

--40 -30

D

,0-oA

-50 -30 -10 +10 +30-40 -20 0 +20

Vm, mV

FIG. 1. Steady-state synaptic currents and conductance change produced by the natural transmitter released after stimulation of thepleuro-abdominal connectives. (A,) Sensory neuron clamped at -50 mV in normal seawater and stepped to -70 mV for 200 msec at 0.1 Hz formeasurement of membrane conductance. Connective stimulation (CS) is followed by inward shift in holding current. (A2) Expanded currentand voltage records before and after connective stimulation. Hyperpolarizing step before nerve stimulation elicits inward current which comesslowly back to the baseline after return to holding potential (a, broken line). The slow return to baseline suggests the development of a voltage-dependent outward current. After connective stimulation, inward current is decreased and comes back to the baseline more quickly after returnto the holding potential (b). (B,) Steady-state current-voltage curve (continuous line) obtained by measuring current at the end of 5-sec depo-larizing and hyperpolarizing clamp steps from holding potential of -50 mV. After connective stimulation, current is shifted inward (dashedline). Measurements in normal seawater (10mM K+). (B2) Protocol as in B, but in a different cell bathed in high-potassium seawater (20mMK+). Curve obtained after connective stimulation (dashed line) and curve before stimulation (continuous line) intersect at about -47 mV, thereversal potential of the synaptic current. (C) Synaptic current calculated by taking the difference between steady-state current-voltage curvesbefore and after connective stimulation. Continuous line, synaptic current in normal seawater; dashed line, synaptic current in 20mM K+ seawater.Currents are calculated from curves in B, and B2- (D) Steady-state current-voltage curves before (continuous line) and after (dashed line) ap-plication of serotonin (the putative facilitating transmitter) in the presence of nickel, a Ca2+ channel blocking agent. Steady-state outward currentwas decreased at all potentials more depolarized than -40 mV.

indicating that the inward shift in the holding current is ac-companied by a decrease in conductance (Fig. 1A2). Thechanges in the resting current and conductance correspond tothe earlier finding that the long-lasting excitatory postsynapticpotential (EPSP) is due to a decrease in membrane conductance(5).A depolarizing decreased-conductance EPSP can be inter-

preted most simply as the synaptic reduction of a steady-stateoutward K+ (or Cl-) current. The synaptic current was unaf-fected by removal of 85% of the extracellular Cl-. We exam-ined the steady-state current-voltage relationship of the slowEPSP in order to find the reversal potential of the synapticcurrent and to analyze its dependence on K+. In normal sea-water, the relationship of the synaptic current to membranepotential is nonlinear. This finding implies that the synapticconductance is voltage-dependent (Fig. 1BI). In 92% of cells(11 of 12), the synaptic current declined to near zero between-50 and -60 mV but did not reverse (Fig. 1 B1 and C). In 8%of cells (1 of 12), reversal occurred between -60 and -70 mV,in reasonable agreement with the reported reversal potentialsfor K+ currents in molluscan neurons (8, 9).We next doubled the external K+ concentration and found

that the current now reversed in 38% of cells (5 of 13; Fig. 1 B2and C) and the average reversal potential was about -50 mV.Although values for the reversal potential are subject to ex-perimental error and only one value is available for the reversalpotential in normal seawater, the shift of the reversal potentialto a more depolarized level and the 5-fold increase in the pro-portion of cells in which the current reversed in a high K+ so-lution indicate that the slow EPSP is largely the result of a sy-naptically mediated decrease in K+ current.

As a control for the possibility that the EPSP is due to the

direct activation of a Ca2+ current (10), we added nickel orcobalt to the bathing solution in a concentration that blocksCa2+ currents. With the Ca2+ channels blocked, serotonin stilldepressed the remaining K+ currents in a manner similar to theactions of connective stimulation in normal seawater (Fig. ID).However, because we have not yet made a quantitative com-parison between the effects of serotonin on the K+ current withand without the Ca2+ channels blocked, we cannot exclude thepossibility that serotonin may also have a small action on Ca2+or calcium-activated K+ current. Nonetheless, the experimentsindicate that the EPSP is predominantly due to a reduction inK+ current and suggest, moreover, that this K+ current mustinvolve channels such as the delayed and the early K+ channelsinstead of or in addition to the calcium-activated K+channel.

Presynaptic Facilitation Is Associated with a Decreasein the Transient K+ Current Elicited by DepolarizingCommands. Serotonin and the natural transmitter released byconnective stimulation also reduce the transient outward cur-rent and increase the net inward current elicited by brief de-polarizing commands from -40 mV to +20 mV designed toreach the approximate voltage level of the peak of the actionpotential (Fig. 2). This reduction in outward current would leadto a slower repolarization of the action potential if the cell werenot clamped (5). But the question remained: Do the changesin these transient currents also result from a decrease in K+currents, as does the long-lasting EPSP, or does the facilitatingtransmitter also produce changes in the Ca2+ current?To answer this question, we isolated the cell bodies of the

sensory neurons by ligation to optimize voltage-clamp control,and we blocked the Na+ and K+ currents by removing externalNa+ and adding the K+ current blocking agents Et4N+ and

v

Neurobiology: Klein and Kandel

A .

0-.L-

Dow

nloa

ded

by g

uest

on

Dec

embe

r 7,

202

0

6914 Neurobiology: Klein and Kandel

A

ZI I I.C I-.r -- - 3mV

+20mV r Ji4 4--/-/- f --:Vy- i-iJ25 nA-45 %o q_ 1mt %mt I-Trial 1 4 10 15 f 16 20 25 31

CS

B Control (1)Depressed (15)Facilitated (20)

10 nA5 msec

FIG. 2. Decrease in transient outward current and increase in net inward current associated with presynaptic facilitation. (A) The presynapticsensory neuron was voltage clamped in high-divalent-cation seawater containing no blocking agents. The EPSP produced by the sensory neuronis recorded in a motor neuron. The presynaptic membrane potential was stepped from -45 mV to +20 mV at 0.1 Hz (lower traces), elicitingtransient inward and outward currents in the sensory neuron (middle traces) and EPSPs in the follower cell (upper traces). With repeated de-polarizing commands, the EPSPs underwent homosynaptic depression in trials 1-15. Connective stimulation (CS) produced facilitation of theEPSPs (top traces) and reduction of the outward current and increase in the net inward current in the presynaptic neuron in trials 16-31 (middletraces). (B) Three traces from A are superimposed to illustrate lack of significant change in currents during synaptic depression (trials 1 and15) and reduction of outward and increase in inward current associated with presynaptic facilitation (trial 20).

4-aminopyridine (9). In three experiments a normal concen-tration of external Ca2+ was used; in six other experiments Ba2+was substituted for Ca2+ to eliminate the calcium-activated K+current (11).

Neurons were briefly stepped to a depolarized membranepotential at 0.1 Hz. An inward current, carried by ca2+ or Ba2+,flowed in response to the depolarizing step. This current de-creased progressively during stimulation (Fig. 3A, left side).

450 mM11 mM10 mMo Ca2+0 Na'

Et4NBa2+ m

4-AP

This decrease in the current through the Ca2+ channels seemsto contribute to the homosynaptic depression of transmitterrelease that underlies habituation (unpublished data).When serotonin, the putative facilitating transmitter, was

added to the bath, there was no change in the holding current,the resting conductance, or the transient inward currents elic-ited by a step depolarization (Fig 3 A and C2). When thesebathing solutions were replaced with normal seawater, serotonin

A

f fi rIu-

Trial 1 2 5 10 12 15 16 20 25 29

,rotonin

B X/ _y $1V<V& l2nA

Vm 235 mV IL L IL SL L ILIL IL IL50 msec

Trial 1 2 5

C,

Im - L ~;J~J 1 nA100 msec

Vm -70_ LV LV

Control Serotonin

10 12 15 20 23 25 30

Serotonin

C2

450mM Et4N+'m 10j.5 nA10 mM 4-AP11 mM Ca2+ 100 msec

0 Na+ Vm -5OmV70 Serotonin

Control Serotonin

FIG. 3. Effects of serotonin on membrane currents in the presence and absence of K+ current blockers. Parts A-C are from sensory neu-

rons isolated by ligation. (A) With K+ currents blocked [with Et4N+, 4-aminopyridine (4-AP), and substitution of Ba2+ for Ca2 ], step depo-larizations at 0.1 Hz (bottom traces) caused gradual decline in inward current (top traces). Addition of serotonin had no effect. (B) With thesame cell as in A, the protocol was repeated after the solution containing the K+ blocking agents had been replaced with normal seawater. Serotoninnow caused a decrease in the outward currents (and an increase in the inward currents) elicited by depolarizing steps as well as an inward shiftin the holding current. (C,) In normal seawater, serotonin caused a decrease in steady-state conductance monitored with hyperpolarizing stepsand an inward movement of the holding current. (C2) With Et4N+ and 4-aminopyridine in the bath, serotonin had no effect on the conductanceor on the holding current.

'PostImPreVm

Vm +40mJ-LJ LJ L L§JLrJLIJEJ LfL

Proc. Natl. Acad. Sci. USA 77 (1980)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 7,

202

0

Proc. Natl. Acad. Sci. USA 77(1980) 6915

again produced changes in the sensory neuron current similarto those usually observed with the natural transmitter releasedby connective stimulation: serotonin decreased the leakagecurrent and the transient outward current and caused theholding current to become more inward (Fig. 3 B and Cl). Theresults of these experiments imply that serotonin and, pre-sumably, the natural transmitter, act by decreasing K+ currentand not by increasing directly the current through the Ca2+channel.

Because the K+ blockers might affect the properties of theCa2+ current or might block the serotonin receptors on the cellmembrane, we carried out additional experiments to test fur-ther the hypothesis that serotonin acts exclusively on K+ current.K+ currents can also be eliminated by replacing the intracellularK+ with impermeant Cs+ after first making the cell permeableto monovalent cations with the antibiotic nystatin (6, 7). Aftertreatment with nystatin and bathing in a solution rich in Cs+and free of K+, the ganglia were placed in a solution containingonly Ca2+, Mg2+, Tris, and Cl-. Neurons were voltage clampedby using 3 M CsCl electrodes to avoid leakage of K+ into thecells from KC1 electrodes. Brief depolarizing commands pro-duced a transient and relatively pure inward Ca2+ current thatcould be blocked with nickel. When serotonin was added to thebath, there was no change in current in each of six experiments[Fig. 4 A (left side) and B].To ensure that these results were due to K+ washout and not

to the nystatin treatment itself, some cells were impaled witha KCI electrode after the washout of K+ and K+ was allowedto leak into the cell. An outward current carried by K+ nowmasked the inward current seen when only CsCl electrodeswere used [Fig. 4A (right side)]. When serotonin was added to

A5-HT, control 5-HT- ~

Control

a -j1 nA .j2nA

V+10 mV, 5 msec +15 10 msec

m.45 -45*

BIm

Control 5-HT

1E (L 10.5 nA"-00 me

Vm -65

C Control 5-HT

ImIm - ; , - .5 nA~~~~~ ~~10 msecVm65

FIG. 4. Effects of serotonin on the currents in the sensory neuronafter washout and partial restoration of intracellular K+. (A) Sensoryneuron clamped with two CsCl electrodes after nystatin treatmentand replacement of intracellular K+with Cs+. Left side shows thatdepolarizing steps at 0.1 Hz elicit an inward current that is due to Ca2+and is blocked by Ni2+. Serotonin (5-HT) had no effect on this cur-rent. The upper trace shows superimposed records of the inwardcurrents before and after application of serotonin. Broken line rep-resents holding current. On right side, in another cell clamped after

nystatin treatment and K+ washout, a KCl electrode was used inconjunction with a CsCl electrode so as to allow K+to leak into thecell. Step depolarizations elicited an outward current carried by K+leaked into the cell from the KC1 electrode. Addition of serotoninreduced the outward current. (B) The steady-state conductance wasunaffected by serotonin in the Cs-perfused cell studied with two CsClelectrodes. (C) The steady-state conductance was-decreased by se-rotonin in a Cs-perfused cell into which K+ was leaked from a KClelectrode.

the bath, this outward current decreased, as did the restingconductance measured with hyperpolarizing pulses (Fig. 4C).These experiments support the hypothesis that serotonin and,presumably, the natural transmitter released by stimulation ofthe connective decrease K+ current and do not act directly onthe Ca2+ channel.Changes in the Shape of Monosynaptic EPSPs Reflect the

Changes in the Presynaptic Currents. Decreasing the K+current activated by an action potential could cause the actionpotential to broaden, allowing greater Ca2+ inflow and givingrise to increased transmitter release. We previously found (5)that the action potentials of sensory neurons do in fact broadenafter connective stimulation. The mean (+ SD) increase induration in six experiments was 10 : 8%. To see whether thesemodest increases in duration could account for the large changesin the size of the monosynaptic EPSPs produced by the sensoryneurons as a result of presynaptic facilitation, we examined therelationship between duration of presynaptic depolarizationand amplitude of the EPSPs. We found that increasing theduration of the step from 20 to 25 msec doubled the size of theEPSPs. The relationship was steeper for shorter pulses than forlonger ones, lending support to the idea that the small increasesin spike duration that occur in drug-free solution could causesignificant synaptic facilitation.When EPSPs elicited with commands of different duration

were normalized to the same amplitude and superimposed, wefound that longer depolarizations gave EPSPs with prolongedrise times and prolonged initial decay times. By contrast, Cas-tellucci and Kandel (12) had found that changing EPSP am-plitude by altering the calcium concentration produced nochange in the shape of the EPSP. Thus, changing the size of theEPSP by changing the duration of the Ca2+ current affects theEPSP differently from changing the EPSP size by changing theamplitude of the current. If presynaptic facilitation is causedby spike broadening, there should be a lengthening of the initialportion of the EPSP after nerve stimulation. We found that,during depression produced by repeated stimulation of thesensory neurons, EPSP amplitude decreased to a fraction of theinitial value with no change in shape. By contrast, after a fa-cilitating stimulus, EPSPs had a prolonged initial phase, evenwhen they were smaller than the EPSPs elicited at the begin-ning of the experiment. These results, found in each of sevenexperiments, confirm predictions that presynaptic facilitationis caused by prolongation of the presynaptic Ca2+ currentsecondary to the spike broadening produced by depression ofK+ current.

DISCUSSIONPresynaptic Facilitation Is Due to a Depression of the K+

Current. Presynaptic facilitation appears to be caused by anincreased inflow of Ca2+ secondary to depression of thepresynaptic K+ current. This hypothesis is supported by threetypes of evidence: (i) facilitation of the monosynaptic EPSPelicited in the follower cells by stimulation of a sensory neuronwas never observed without a concomitant decrease in K+current; (ii) in all seven experiments in which EPSPs wereelicited with action potentials in the sensory neurons withoutchannel blocking agents present, presynaptic facilitation wasaccompanied by prolongation of the initial phase of the EPSP;and (iii) varying the duration of the presynaptic depolarizationunder voltage clamp suggests that the action potential broad-ening that occurs after nerve stimulation may be adequate toaccount for presynaptic facilitation.We have assumed that the changes in the steady-state current

and in the currents elicited with depolarizing command stepsare aspects of a single phenomenon. The findings that they both

Neurobiology: Klein and Kandel

Dow

nloa

ded

by g

uest

on

Dec

embe

r 7,

202

0

6916 Neurobiology: Klein and Kandel

Na' channel

Ca" channel(open)

Ca" channel ° 0

(cloned)---;o~ o

0 0 0000 0a

0 0

Venicle0 0.

K channel *(open) ;

Canr

Control Sensitization

Postsynaptic cell

FIG. 5. Molecular model of presynaptic facilitation and sensiti-zation. (A) Modulatory neurons release serotonin which increases thelevel ofcAMP in the terminals of the sensory neuron (large triangles).The cAMP acts on voltage-sensitive K+ channels to decrease the K+current. This prolongs the action potential, increases the Ca2+ influx,and thereby augments transmitter release by allowing more synapticvesicles to bind to release sites, a prerequisite step for exocytotic re-lease of transmitter. Arrows from K+ to Ca2+ channels indicate flowof hyperpolarizing current through the K+ channels. This K+ currentturns off the Ca2+ current and prevents additional Ca2+ channels fromopening. (B) Postulated biochemical steps in the action of serotonin(5-HT) on K+ channels involving a cAMP-dependent protein kinase.See text for details. Ad. Cy., adenyl cyclase.

occur in response to nerve stimulation, have similar timecourses, and are both blocked by K+ blocking agents and Cs+substitution support this assumption. Nevertheless, since thisquestion has not been examined adequately, it is possible thatthe two effects reflect distinct K+ channels. Indeed, the quan-titative contribution of the three known K+ channels to the K+current depression has not yet been explored.

Depression of the K+ Currents May Involve the Action ofa cAMP-Dependent Protein Kinase. There is reason to believethat ion channels are integral membrane proteins (13). As a

result, the ability to specify the particular membrane channelsinvolved in presynaptic facilitation is an essential first steptoward a biochemical analysis of the sequence of molecularmechanisms initiated by the facilitating transmitter. Indeed,the available data allow a specific, albeit tentative, molecularmodel to be considered (Fig. 5). The evidence suggests thatserotonin is the likely transmitter for presynaptic facilitation(2, 5, 14, 15). Serotonin stimulates the synthesis of cAMP (4),and both serotonin and cAMP in turn have been shown to en-hance transmitter release (2) and to increase the Ca2+ currentin the cell body (5) by depressing the opposing K+ current.Following the findings by Krebs and his colleagues (16) thatcAMP stimulates glycolysis in muscle by means of a cAMP-dependent protein kinase, Kuo and Greengard (17) suggestedthat all actions of cAMP are mediated by this family of en-zymes. It therefore is attractive to think that the serotonin-stimulated increase in cAMP enhances the activity of acAMP-dependent protein kinase in the presynaptic terminalsof the sensory neurons and that the kinase phosphorylates amembrane related protein, perhaps the K+ channels themselves,thereby closing the K+ channels (Fig. 5). This model thereforepredicts that intracellular injection of the catalytic unit of thecAMP-dependent protein kinase would decrease the K+ cur-rents and simulate presynaptic facilitation and that serotonin,the putative facilitating transmitter, would cause phosphoryl-ation of a membrane protein that is specifically related to theK+ channel (18).

This work was supported by a Career Scientist Award (5-KO5-MH-18558) to E.R.K. and by grants from the National Institutes ofHealth (GM-23540 and MH26212) and the McKnight Foundation.

1. Castellucci, V. & Kandel, E. R. (1976) Science 194, 1176-1178.

2. Brunelli, M., Castellucci, V. & Kandel, E. R. (1976) Science 194,1178-1181.

3. Cedar, H., Kandel, E. R. & Schwartz, J. H. (1972) J. Gen. Physiol.60,558-569.

4. Cedar, H. & Schwartz, J. H. (1972) J. Gen. Physiol. 60, 570-587.

5. Klein, M. & Kandel, E. R. (1978) Proc. Nati. Acad. Sci. USA 75,3512-516.

6. Russell, J. M., Eaton, D. C. & Brodwick, M. S. (1977) J. Membr.Biol. 37, 137-156.

7. Tillotson, D. & Horn, R. (1978) Nature (London) 273, 312-314.

8. Meech, R. W. & Standen, N. B. (1975) J. Physiol. (London) 249,211-239.

9. Thompson, S. H. (1977) J. Physiol. (London) 265, 465-488.10. Pellmar, T. C. & Carpenter, D. 0. (1979) Nature (London) 277,

483-484.11. Connor, J. A. (1979) J. Physiol. (London) 286,41-60.12. Castellucci, V. & Kandel, E. R. (1974) Proc. Natl. Acad. Sci. USA

71,5004-5008.13. Finkelstein, A. & Mauro, A. (1977) Physical Principles and

Formalisrms of Electrical Excitability, Handbook of Physiology,ed. Kandel, E. R. (American Physiological Society, Bethesda,MD), Vol. 1, Part 1, pp. 161-213.

14. Tomosky-Sykes, T. K. (1978) Soc. Neurosci. 4, 208 (abstr.).15. Bailey, C. H., Hawkins, R. D., Chen, M. C. & Kandel, E. R. (1980)

J. Neurophysiol., in press.16. Walsh, D. A., Perkins, J. P. & Krebs, E. G. (1968) J. Biol. Chem.

243,3763-3765.17. Kuo, J. F. & Greengard, P. (1969) Proc. Natl. Acad. Sci. USA 64,

1349-1355.18. Paris, C. G., Kandel, E. R. & Schwartz, J. H. (1980) Soc. Neurosci.

6, 844 (abstr.).

Proc. Natl. Acad. Sci. USA 77 (1980)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 7,

202

0