Douglas A. Baxter and John H. Byrne- Serotonergic Modulation of Two Potassium Currents in the...

8/3/2019 Douglas A. Baxter and John H. Byrne- Serotonergic Modulation of Two Potassium Currents in the Pleural Sensory Neurons of Aplysia

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JOURNAL. OF NEUROPHYSIOLOC;~Vol. 62. No. 3. September 1989. Prruied IH L..S..d.

Serotonergic Modulation of Two Potassium Currentsin the Pleural Sensory Neurons of AplysiaDOUGLAS A. BAXTER AND JOHN H. BYRNEDepartment ~fNeurohio/ogv and Anatomy, The University of Texas Medical School, Houston, Texas 77225SUMMARY AND CONCLUSIONS

1. The properties of membrane currents that were modulatedbv serotonin (5-HT) were investigated with two-electrode vol t-age-clamp techniques in sensory neuron somata isolated from thepleural ganglion of Ap/evsia cal(fh-nica. The modulatory eff ect s of5-HT were revealed by computer subtraction of current responseselicited in the presence of 5-HT from current responses elicitedprior to the application of 5-HT. The complexities of the resulting5-HT difference currents (J5-& suggested that 5-HT modulatedmore than one component of membrane current.2. The 5-HT difference currents appeared to have at least twodistinct components. One component was clearly evident atmembrane potentials more negative than - 10 mV was relativelyvoltage independent and did not inactivate. A second componentwas activated at membrane potentials more positive than - 10mV, had complex kinetics, and was highly voltage dependent. Inan attempt to iden tify the membrane currents that were modu-lated by 5-HT, we compared the pharmacologic sens itiv ity ofZSeHT.o that of previous ly described K+ currents.3. The two components of 15-H1 had different sensitivi ties toagents that block KS currents. The relatively voltage-independentcomponent of IS-H7- was not blocked by 2 mM 4-aminopyridine(4-AP) and was relatively insensitive to tetraethylammonium(TEA) (estimated Kd of 92 mM). In contrast, the voltage-depen-dent component of 15-HT.was blocked by 4-AP (2 mM) and mod-erate concentrations of TEA (estimated Kd of 5 mM).4. The KS current blockers that were used to examine 15-HTwere also used to examine voltage-activated membrane currents.Externally applied TEA blocked the delayed or voltage-dependentKS current (IK r-) with an estimated dissociation constant (&) of 8mM and a membrane current similar to the Ca2+-activated K+current (IK,& with an estimated Kd of 0.4 mM. In addition, ex-ternally applied 4-AP (2 mM) blocked IKqI . Thus TEA and 4-APwere equipotent in blocking both IK.l. and the voltage-dependentcomponent of 15-f11..5. The suggestion that ls-l-l.l. contained multiple componentswas supported further by examining the modulatory eff ect s ofadenosine 3,5-cyclic monophosphate (CAMP) that mediatessome actions of 5-HT on membrane currents in these cells. CAMPdifference currents (IcAMP) were similar to the relatively voltage-independent component of JSeHT. The subsequent addition of5-HT to solutions already containing CAMP resulted in 5-HTdifference currents similar to the voltage-dependent componentof 15-H,T.Thus the voltage-dependent component of 15+,T did notappear to be modulated by elevated levels of CAMP.6. Similarly, the modulatory eff ect s of small cardioactive pep-tide (SCPb) that mimics 5-HT and elevates levels of CAMP re-vealed difference currents (I s(Ph) that were similar to both IcAMPand the relatively voltage-independent component of ]5-HT. Thesubsequent addition of 5-HT to solutions already containingSCPh resulted in 5-HT difference currents similar to the voltage-dependent component of JSmHT.. hus SCPb did not appear tomodulate a current similar to the voltage-dependent componentof &-1,-l *

7. Based on pharmacologic sens itiv ity and voltage dependence,we conclude that in somata of pleural sensory neurons brief de-polarizing voltage-clamp pulses activate at least two currents thatcould be modulated by 5-HT. The relative ly voltage-independentcomponent had properties consistent with the previously de-scribed S current. The voltage-dependent component had proper-ties similar to IK,L,.

INTRODUCTION

In Aplysia cafifornica, cellular mechanisms underlyingneuronal plasticity have been analyzed extensively in twopopulations of sensory neurons. One population is locatedin the abdominal ganglion and mediates the siphon-gillwithdrawal reflex. The second population is located in thepleural ganglion and mediates the tail withdrawal reflex.Many analyses of the biophysical mechanisms of plasticityin these sensory neurons have focused on a serotonin(5-HT)-sensitive K+ current, which is termed the S current(Klein et al. 1982; Pollock et al. 1985). Extracellular appli-cation of 5-HT reduces the magnitude of the S current viaCAMP-dependent protein phosphorylation that closes thechannel (Klein et al. 1982; Siegelbaum et al. 1982, 1987;Camardo et al. 1983; Pollock et al. 1985, 1987; Shuster etal. 1985; Belardetti and Siegelbaum 1988). The S currentcontributes to the resting K+ conductance and to the repo-larization of action potentials in these cells. The serotoner-gic modulation of the S current is postulated to be an im-portant mechanism contributing to presynaptic facilitationof transmitter release from sensory neurons, which in turnis thought to be the cellular basis of several simple forms oflearning (for reviews see Byrne 1985, 1987; Carew et al.1986, 1987; Hawkins et al. 1986; Kandel and Schwartz1982).Several criteria are used to distinguish the S current fromother KS currents that are found in molluscan neurons(e.g., Thompson 1977; Adams et al. 1980a; Byrne 1980),including its voltage dependence, kinetics, and pharmaco-logic sensitivity. The S current is relatively voltage indepen-dent, active over a wide range of membrane potentials,noninactivating, increases in a time-dependent mannerduring depolarization, and is relatively insensitive to theK+ channel blockers 4-aminopyridine (4-AP) and tetraeth-ylammonium (TEA) (Klein et al. 1980, 1982; Paupardin-Tritsch et al. 198 1; Siegelbaum et al. 1982, 1987; Camardoet al. 1983; Walsh and Byrne 1984, 1989; Pollock et al.1985, 1987; Shuster and Siegelbaum 1987; Scholz andByrne 1987, 1988; Brezina et al. 1987, 1988). More recent

0022-3077/89 $1 SO Copyright 0 1989 Th e American Physiological Society 665


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MODULATION OF IS+ CURRENTS 667wereestimated rom previously reported dose-responseelation-ships Thompson 1977; Hermann and Gorman 198 a,b; Stan-field 1983; Ocorr and Byrne 1985; Deitmer and Eckert 1985;Shusterand Siegelbaum1987; Brezina et al. 1987, 1988; Rudy1988) and independently confirmed during the courseof theseexperiments.The concentrationsand the method of bath appli-cation are similar to those used n other studieson the sensoryneurons (e.g., Klein et al. 1980, 1982, 1986; Ocorr and Byrne1985,1986;Pollock et al. 1985;Hochner et al. 1986b;ShusterandSiegelbaum 987;Walshand Byrne 1989).Recordings rom cellswith the following characteristicswereincluded in the data set: 1) stable resting membrane conduc-tances,2) membranecurrents hat did not showprogressive, on-specificalterationsduring repetitive voltage-clamppulses, ) rest-ing membranepotentials greater than -40 mV [-43 t 3 mV(mean SD)], and 4) input resistancesreater han 15 MQ (32 t8). The resultsof this study representsuccessfulecordings rom140preparations.RESULTSModulatory eflects of 5-HT on membrane currents

We examined the modulatory effects of 5-HT on mem-brane currents by isolating 5-HT difference currents (&&.An example of the method used to isolate 15-HT s shown in

1 Step to -20 mV

J 5 nA20 msecA2 5-HT Difference Current (a - b)

-J 2 nA20 msec

Fig. 1. The membrane currents that were elicited in ASWby a voltage-clamp pulse from -90 to -20 mV are shownin Fig. IA1 (trace a). The addition of 5-HT (30 PM) re-duced the total membrane current (trace b). This reductionis particularly evident at the end of the voltage-clamppulse. The difference between the current responses elicitedin the presence of 5-HT (trace b) and the current responseselicited in ASW (trace a) represents the membrane currentthat was modulated by 5-HT. Computer subtraction of the5-HT trace from the ASW trace results in a 5-HT differ-ence current, I 5-n-r Fig. lA2), in which the 5-HT-mediatedreduction in outward current is represented as an upwarddeflection. Previous work indicates 5-HT reduces outwardmembrane current, at least in part, by initiating the closureof a distinct class of 5-HT sensitive K+ channels, the Schannels (Klein et al. 1982; Siegelbaum et al. 1982, 1987;Camardo et al. 1983; Pollock et al. 1985, 1987). Thus theexample of I 5-H-rshown in Fig. IA2 represents the S currentand has properties consistent with those previously re-ported for the S current (see also below).In contrast to the relatively simple modulation of mem-brane currents by 5-HT shown in Fig. IA, a complex 5-HTdifference current was observed at more depolarized mem-

Bl Step to +20 mV

b + 5-HTa ASW

I 30 nA20 msecB2 5-HT Difference Current (a - b)

FIG. 1. Modulation of membrane currents by 5-HT at 2 different levels of depolarization. Al: membrane currents wereelicited by voltage-clamp pulses from -90 to -20 mV in ASW (trace a) and following bath application of 5-HT (30 PM)(trace b). Note that the membrane currents do not return to the preclamp level since the cell was clamped from a holdingpotential of -90 to -20 mV and then back to -50 mV (see METHODS). A2: the 5-HT difference current (I,-,,) was isolated bysubtracting the current response elicited in the presence of 5-HT from the current response elicited in ASW (a - b). BI:membrane currents from the same cell were elicited by voltage-clamp pulses from -90 to +20 mV in ASW (trace a) andfollowing bath application of 5-HT (30 PM) (trace b). B2: 5-HT difference current (I 5+rr) was isolated by subtracting thecurrent response elicited in the presence of 5-HT from the current response elicited in ASW (a - b). Note changes inordinate scales. The complexity of the waveform of the 5-HT difference current obtained at +20 mV indicates that 5-HT ismodulating not only the S current but also some other component of membrane current.


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668 D. A. BAXTE R AND J. H. BYRNEbrane potentials. The membrane current that was elicitedin ASW by a voltage-clamp pulse from -90 to +20 mV areshown in Fig. lB2 (trace a). Application of 5-HT (30 PM)appeared to slow the rate-of-rise of the total membranecurrent and thus markedly decreased the total current earlyduring the voltage-clamp pulse. In addition, 5-HT ap-peared to slow the inactivation of the outward membranecurrent and thus increased the outward current later duringthe voltage-clamp pulse. The difference between the cur-rent responses elicited in the presence of 5-HT and thecurrent responses elicited in ASW represents the modula-tory effects of 5-HT at more depolarized levels and isshown in Fig. lB2. The early upward peak of 15-HT in Fig.lB2 reflects the initial decrease in the total membrane cur-rent (Fig. 1BI), whereas, the downward shift in Fig. lB2represents the late increase in the total membrane currentat the end of the voltage-clamp pulse after application of5-HT (Fig. 1BI). A comparison of the 5-HT differencecurrents shown in Fig. 1, A2 and B2 indicates that the

Al ASW Bl ASW + 5-HT

modulatory effects of 5-HT are more complex than simplyinitiating the closure of the S channel and that other mem-brane currents are involved.A complete series of 5-HT difference currents that wereisolated from a wide range of voltage-clamp potentials isshown in Fig. 2. The membrane currents that were elicitedin ASW by voltage-clamp pulses from -90 to -30, -20,and - 10 mV are shown in A2 and the membrane currentsthat were elicited by voltage-clamp pulses to 0, + 10, and+20 mV are shown in AI (note the change in gain). Themembrane currents that were elicited by identical voltage-clamp pulses following application of 5-HT are shown inFig. 2B. The 5-HT difference currents that were isolated bysubtracting the current responses elicited in the presence of5-HT from the current responses elicited in ASW areshown in Fig. 2C. At membrane potentials more negativethan 0 mV (Fig. 2C2), I 5-HT increased slowly during thevoltage-clamp pulses, and the peak amplitudes increasednearly linearly with voltage (also see Fig. 8). This relatively

20 msecB2

-10 mV

-20-30

J 5 nA20 msecC 1 5-HT Difference Currents (A - B)

0 mV -I 15 nA20 msec

FI G. 2. Voltage sensitivity of currents mo dulated by5-HT. Membrane currents were elicited by voltage-clamp pu lses from -90 mV to membrane potentialsranging between -30 and +20 mV. A: current response swere elicited in ASW. S mall currents elicited with sma lldepolarizations are shown in A2, whereas large currentselicited by larger depolarizations are shown in A 1. B:current response s to potentials corresponding to thoseshown in Al and A2 were e licited in the same cell follow-ing bath app lication of 5-HT (30 FM). C: 5-HT differ-ence currents were isolated by subtracting the currentresponse s elicited in the presence of 5-HT from the cor-responding current responses elicited in ASW (A - B).Resu lts s imilar to these were obtained in all preparations(n = 33) in which I 5-Hsr was isolated from neurons bathedin ASW. Note changes in ordinate scales. The compo-nent of &-HT expressed at sma ll depolarizations (c2) ex-hibits little voltage-dependence and has simple kinetics,whereas the compone nt expressed at larger depolariza-tions (CI) is highly voltage dependent and has complexkinetics.

-20-30 J 2 nA

20 msec


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MODULATION OF K CURRENTS 669

voltage-independent component of ZsmHT id not inactivateduring the 200 ms depolarizing voltage-clamp pulses.Moreover, it did not inactivate during long-duration (1min) voltage-clamp pulses to -20 mV (n = 9; data notshown). These properties are consistent with the previousdescriptions of S current (Klein et al. 1980, 1982; Siegel-baum et al. 1982, 1987; Camardo et al. 1983; Walsh andByrne 1984, 1989; Ocorr and Byrne 1985; Pollock et al.1985, 1987: Scholz and Byrne 1987, 1988). In contrast, themagnitude, rate of activation, and rate of inactivation of5-HT difference currents that were isolated from voltage-clamp pulses to membrane potentials more positive than- 10 mV were highly voltage dependent (Fig. 2Cl). Thecharacteristics of lsmH7at depolarized membrane potentials(Fig. 2Cl) are inconsistent with the those of the S current.These results suggest that 5-HT modulates an additionalvoltage-dependent membrane conductance that is acti-vated at membrane potentials more positive than - 10 mV.Separation c!fK currents

While the relatively voltage-independent component ofthe 15-H7-has properties similar to the S current, the volt-age-dependent component of Ir+.r has not been describedpreviously. We were interested, therefore, in determiningwhether this second component represented the modula-tion bv 5-HT of either the voltage-dependent IS+ current(Ik ,-),?a+-activated K current (ZKCa), or transient IScurrent (I,), which are found in these sensory neurons(Klein et al. 1982; Pollock et al. 1985; Baxter and Byrne1988: Walsh and Bvrne 1989) and other molluscanneurons (e.g., Connorand Stevens 197 1; Thompson 1977;Aldrich et al. 1979; Adams et al. 1YSOa,b; Byrne 1980:Gorman and Thomas 1980; Hermann et al. 198 I a,b, 1983;Hockberger and Connor 1984; Strong et al. 1984, 1986;Thomas 1984; Deitmer and Eckert 1985; Junge 1985;Kehoe 1985: Bezanilla et al. 1986: Acosta-Urquidi 1988:also see, Hille 1984; Latorre et al. 1984; Rogawski 1985;Rudy 1988). The method that we chose to investigate thispossibility was to compare the pharmacologic sensitivity,voltage-dependence, and kinetics of the voltage-dependentcomponent of 15-H-r to those of Ik I., Jk Ca, and I*. An ex-tensive body of literature indicates that& I,, and IKCa canbe blocked by the use of appropriate concentrations ofTEA and 4-AP and that IA can be selectively inactivated atrather hyperpolarized membrane potentials (e.g., Connorand Stevens 197 1; Thompson 1977; Byrne 1980; Hermannet al. 198 la,b, 1983; Klein et al. 1982; Stanfield 1983; Hille1984; Latorre et al. 1984; Rogawski 1985; Blatz and Mag-leby 1987; Shuster and Siegelbaum 1987; Cook 1988:Rudy 1988: Walsh and Byrne 1989). Therefore, we usedthese methods to isolate examples of these three classes ofKt currents and compare their characteristics with thoseot I>-HT-I- k.I.* In previously studied neurons of Aplwia, TEA hasbeen reported to block IK 1vwith an apparent dissociationconstant (Kd) of 6-8 mM and some examples of 1k (Jawith arcb near 0.5 mM (Thompson 1977; Byrne 1980; Hermannand Gorman 198 1b; Klein et al. 1982; Deitmer and Eckert1985; Walsh and Byrne 1989). Thus low concentrations ofTEA should block 1kCa significantly but have little effect

on IK,Ir. Subsequent increases in the bath concentration ofTEA should block the remaining 1k v. Consequently, weexamined the membrane currents that are sensitive to lowand high concentrations of TEA. The membrane currentsthat were el icited by voltage-clamp pulses to +20 mV firstin ASW, following addition of 2 mM TEA, and followingthe increase of the concentration of TEA to 50 mM areshown in Fig. 3Al. Although the low concentration of TEAreduced the membrane current at the end of the voltage-clamp pulse, the low concentration of TEA had little effecton the peak amplitude of the current response. Increasingthe concentration of TEA to 50 mM markedly reduced thepeak amplitude of the current response. The membranecurrents that were blocked by low concentrations of TEAwere isolated by subtracting current responses elicited byvoltage-clamp pulses to 0, + 10, and +20 mV in the pres-ence of 2 mM TEA from corresponding current responseselic ited in ASW (Fig. 3A2). The current affected by lowconcentrations of TEA was slow to activate, showed l ittleinactivation, and had a mild voltage dependence. In theserespects, it bears some resemblance to the S current and the5-HT difference currents illustrated in Fig. 2C2. It differsfrom 15-H-r observed at small depolarizations, however, inits threshold for activation and sensitivity to TEA (seebelow). The sensitivity to low concentrations of TEA sug-gests that the difference currents in Fig. 3A2 may representa TEA-sensitive form of 1k Ca. The membrane currents thatwere blocked by high concentrations of TEA were isolatedby subtracting the current responses elic ited by voltage-clamp pulses to 0, + 10, and +20 mV in high concentra-tions of TEA from corresponding current responses elic itedin low concentrations of TEA (Fig. 3A3). The current af-fected by high concentrations of TEA began to be activatedat potentials more positive than - 10 mV, and both itsactivation and inactivation kinetics are voltage dependent.The large magnitude of the high TEA difference current,the marked voltage dependence of its peak ampl itude andthe voltage dependence of its kinetics of activation andinact ivation are similar to those reported for & in thesesensory neurons and other Apl-ysia neurons (Thompson1977; Aldrich et al. 1979; Adams and Gage 1980a; Byrne1980; Hermann and Gorman 198 1b; Klein et al. 1982;Pollock et al. 1985; Strong and Kaczmarek 1986).In certain neurons of Aplvsia, low concentrations of4-AP have been reported to block Ik I, with a & of -0.8mM, while having no effect on lk.Ca (Hermann and Gor-man 198 la; also see Rudy 1988). Thus we examined themembrane currents that are sensitive to low concentrationsof 4-AP. An example of the membrane currents that wereelic ited first by a voltage-clamp pulse to +20 mV in ASWand then with an identical depolarizing step after applying1.5 mM 4-AP are shown in Fig. 3BZ. The 4-AP markedlyreduced the peak ampl itude of the current response buthad litt le effect on the current at the end of the voltage-clamp pulse. The membrane currents that were blocked bylow concentrations of 4-AP were isolated by subtractingcurrent responses elic ited by voltage-clamp pulses to 0,+ 10, and +20 mV in the presence of 4-AP from corre-sponding current responses elicited in ASW (Fig. 3B2).These 4-AP difference currents are similar to the high TEAdifference current (Fig. 3A3) and have characteristics con-sistent with those of 1k I.


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D. A. BAXTE R AND J. H. BYRNE

TEAA 1 Step to +20 mV

+ 2 mM TEA+ 50 mM TEA

J 20 nA20 msecA2 Low TEA Dif ference Currents (a - b)

Bl Step to +20 mV

B2 4-AP Difference Currents (a - b)/mvi OnA0Zec

A3 High TEA Difference Currents (b - c)nA

FIG. 3. Pharma cologic sensitivity of I k.1.. A 1: currents were elicited by a voltage-clamp pulse from - 50 to +20 mV inASW (trace a), following the addition of 2 mM T EA (trace b), and following the increase of the concentration of TEA to 50mM (trace c). A2: TEA difference currents in this cell were isolated from voltage-clamp pulse s to 0, + 10, and +20 m V. Thecompone nt of membrane current that was blocked by the low concentration of TEA was isolated by subtracting currentresponse s elicited in the presence of 2 mM TEA from co rresponding current responses elicited in ASW (e.g., a - b in AI).A3: the componen t of membrane current that was blocked by higher conce ntrations of TEA was isolated by subtractingcurrent responses elicited in the presence of 50 mM TEA from corresponding current responses elicited in the presence of 2mM TEA (e.g., b - c in Al). Th ese TE A difference currents are representative of results in 14 different preparations. BI: in adifferent cell, membrane currents were elicited by voltage-clamp pulse s from -50 mV (a holding potential that inactivatedIA, e.g., see Fig. 5A) to +20 m V in ASW (trace a) and following the addition of 1.5 mM 4-AP to the bath (trace b). B2: 4-APdifference currents were isolated from voltage-clamp pulses to 0, + 10, and +20 mV by subtracting current response s e licitedin the presence of 4-AP from corresponding current response s elicited in ASW (e.g., a - b in Bl). These difference currentsare representative of results obtained in 12 different preparations. The currents blocked by high concen trations of TEA (50mM, A3) and low concen trations of 4-AP (1.5 mM, B2) are nearly identica l in their voltage se nsitivity and kinetics ofactivation and inactivation. Thes e features are characteristic of IK,I.

pica. Walsh and Byrne (1989) have shown that in thesesensory neurons low concentrations of TEA (5 mM) block This result indicates that an additional component ofmembrane current can be blocked by low concentrationsa K current that is activated by intracellular injection ofCa2+ (also see, Hermann et al. 198 b, 1983; Klein et al. of TEA even after a significant component of membranecurrent has been blocked already by 4-AP. This pharmaco-1982; Stanfield 1983; Hille 1984; Thomas 1984; Deitmer logic sensitivity is consistent with TEA-sensitive forms ofand Eckert 1985; Kehoe 1985; Sawada et al. 1987, 1989). Ik Ca. The membrane current that is blocked by low TEAAs shown in Fig. 3, externally applied 4-AP (2 mM) blocks (bht not by 4-AP) was isolated by subtracting current re-I K 2,,but apparently not the low TEA difference current,which resembles Ik,ca sponseselicited by voltage-clamp pulses o 0, + 10, and +20(Thompson 1977; Hermann et al. mV in the presence of low concentrations of TEA (plus198 a, 1983). Thus we examined the membrane currents 4-AP) from corresponding current responses elicited inthat were insensitive to 4-AP but were sensitive to TEA. ASW that contained 4-AP (Fig. 4B). The low TEA differ-Membrane currents were elicited by a voltage-clamp pulse ence currents became significantly activated at membraneto +20 mV first in ASW containing 4-AP, following the potentials more positive than - 10 mV and were slow toaddition of 2 mM TEA, and following the increase of the develop. With voltage-clamp pulses above +30 mV, theconcentration of TEA to 50 mM (Fig. 4A). Application of a amplitude of the low TEA difference currents began tolow concentration of TEA markedly reduced the mem- decrease data not shown). These low TEA difference cur-brane current. Increasing the concentration of TEA to 50 rents and those in Fig. 3A2 appear to be Zk Ca,based on themM had little additional effect on the membrane currents. voltage dependence and kinetics of this current and its


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MODULATION OF K+ CURRENTS 671

A Step to +20 mVa ASW+ 1.5mM4-AP

b +2 mMTEAc + 50 mM TEA.

J 20 nA20 msec -

B Low TEA Difference Currents (a - b)

15nA20 msec

20 mV

FIG. 4. Effects of TEA on membrane currents after preexposure to4-AP. A: membrane currents were elicited by a voltage-clamp pulse from-70 to +20 mV in ASW containing 1.5 mM 4-AP (trace a) to block IKql.(see Fig. 3B), in the presence of 2 mM TEA (trace b) to block TEA-sen si-ti ve IK,Ca T and in the presence of 50 mM TEA (trace c) to block anyresidual IK, IJ or TEA-sen sitive IK,Ca. Applica tion of a low concentration ofTEA markedly reduced the membrane current. Increasing the concentra-tion of TEA to 50 mM had little additional effect o n the membranecurrents. B: low TEA difference currents were isolated from voltage-clamppulse s to 0, + 10, and +20 mV by subtracting the current response s elicitedin the presence of 2 mM TEA (and 1.5 mM 4-AP) from the correspondingcurrent response s elicited in ASW containing 1.5 mM 4-AP (e.g., a - b inA). Thes e difference currents are representative of results obtained in 13different preparations. The currents blocked by low concen trations ofTEA (2 mM) are slow to activate, do not inactivate, and are mildly volt-age-dependent. Thes e features are charac teristic of ZK,ca.

similarity to IK,Caobserved in these sensory neurons andother neurons of Aplysia (Gorman and Thomas 1980;Hermann et al. 198 1a,b, 1983; Klein et al. 1982; Walsh andByrne 1989). Thus the slow development of the low TEAdifference currents (Figs. 3A2 and 4B) probably reflects thetime course of the accumulation of intracellular Ca2+(Gorman and Thomas 1980). The decrease in peak ampli-tude with large depolarizations is due presumably to thefact that as the membrane potential approaches the Ca2+equilibrium potential, the Ca2 current decreases and lessintracellular Ca2+ is available to activate Ik,Ca (Hermannand Hartung 1983).I~. The membrane currents that were elicited by voltage-clamp pulses to -20 mV from holding potentials of -50and -90 mV are shown in Fig. 5A. The fast, transientcomponent of the outward membrane current that waspresent during the voltage-clamp pulse from -90 mV wasinactivated by the -50 mV holding potential. This com-plete inactivation at relatively hyperpolarized membrane

potentials is a distinctive characteristic of IA (Connor andStevens 197 1; Thompson 1977; Adams et al. 1980b; Byrne1980; Klein et al. 1982; Hille 1984; Latorre et al. 1984;Strong 1984; Junge 1985; Pollock et al. 1985; Rogawski1985; Acosta-Urquidi 1988; Baxter and Byrne 1988; Rudy1988). Thus IA can be isolated by subtracting current re-sponses elicited from a holding potential of -50 mV fromcorresponding current responses elicited from a holdingpotential of -90 mV (Fig. 5B).Three lines of evidence indicate that modulation of IA by5-HT cannot account for the voltage-dependent compo-nent of JS-nT. First, the voltage-dependent component of15-HT s present only at membrane potentials more positivethat - 10 mV (Fig. ZCl), whereas IA is activated by voltage-clamp pulses to membrane potentials more negative than- 10 mV (e.g., Fig. 5B). Second, in sharp contrast to thevoltage dependence of I k,C/ (Fig. 3) and the voltage-depen-dent component of I 5-IIT (Fig. 2C1), the rates of activationand inactivation of IA are relatively voltage independent.Third, the amplitude of IA increases relatively linearly withvoltage (Fig. 5B), whereas both Ik band the voltage-depen-dent Component of&T do not.

A Step to -20 mV

-50 mV-90 mV 2 nA

20 msB Isolated I K A,-10 mV

-20 mV-30 mv

2 nA20 msFIG. 5. Isolation of IA. A: examples of membrane currents elicited by

voltage-clamp pulses to -20 mV from holding potentials of -50 and -90mV . B: compon ent of outward current that was highly sens itive to theholding potential was isolated by subtracting current response s elicited byvoltage-clamp pulses to -30, -20, and - 10 mV from a holding potential-50 mV from the corresponding current response s elicited by depolariza-tions to the same levels but from a holding potential of -90 mV. Thiscurrent is relatively voltage independen t with respect to both its kinetics ofactivation and its kinetics of inactivation. The kinetics of activation andinactivation are rapid compared to both lk,,. (e.g., Fig. 3, A.? and B2) andI 1Ca (Fig. 4B). Thes e features are charac teristic of -I,, . Thes e re sults ind i-cate that the properties of IA are different from those of the voltage-de-pendent compon ent of I 5-HTT and thus, it is unlikely that modulation of 1Aby 5-HT could count for the voltage-dependent compone nt of IwHT.


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672 D. A. BAXTE R AND J. H. BYRNE

Phurmacologic sensitivity of5-HT difference currents.While other interpretations are possible, the similaritybetween the voltage dependence and the complex kineticsof IK,L. and those of the voltage-dependent component ofIsmHT suggested to us that this component of IsmHT may bedue to a modulation of 1k rjby 5-HT. It appeared that 5-HTslowed the activation and inact ivation of Ik v (e.g., Fig.lsl>. Such a modulation would explain both the init ialdecrease and the later increase in the outward membranecurrent induced by of 5-HT. Alternatively, the late increasein membrane current may represent an indirect modula-tion Of 1~ Ca by 5-HT. Boyle et al. (1984) and Spira et al.(1987) found that applicat ion of 5-HT increased the con-centration of intracellular free Ca2+ as measured by theCa2+ indicators arsenazo III and fura-2. These results raisethe possibility that 5-HT could lead to increased activation

Of IK.Ca by increasing the levels of intracellular Ca2+ (seealso Sacktor et al. 1986; Braha et al. 1988). Thus the lateincrease in outward current induced by 5-HT (Fig. IBI)could be due to an increase in 1k,ca. To distinguish between

Al Low TEAa ASW

c (ASW + TEA )b + 2 mM TEA

J 25 nA20 msec

A2 5-HT Difference Current (b - C)

+ 5-HT

these two possibilities, we examined the sensitivity of &-ijTto agents that differentially affect Ik v and 1k,ca.As mentioned above (Figs. 3A and 4), Ik I/ and someforms Of 1~ Ca can be distinguished by their dikerent sensi-tivities to TEA; I k,Cacan be blocked by low concentrationsof TEA that have little effect on IK,c. As shown in Fig. 6A,we examined the sensitivity of the voltage-dependent com-ponent of 15u-r to low concentrations of TEA by isolating15-H-rn solutions containing 2 mM of TEA. Membranecurrents were elicited by voltage-clamp pulses o +20 mV;first in ASW, following the addition of 2 mM TEA andfollowing addition of 5-HT to the solution already con-taining TEA (Fig. 6Al). Despite the presence of a low con-centration of TEA, I 5-H-rstill displayed the characteristicvoltage-dependent component (Fig. 6A2). Thus a concen-tration TEA that blocks an Ik c,-like current (but not Ik v)does not block the voltage-dependent component of &-iT.(A more complete analysis of the sensitivity of &-HT to TEAis presented in Figs. 7 and 9.)IK,v and 1k,Caalso can be distinguished on the basis oftheir sensitivity to 4-AP; Ik v is blocked by low concentra-Bl 4-AP

a ASWb + 1.5mM4-APc (ASW + 4-AP) + 5-HT

I 20 nAB2 5-HT Difference Current (b - c)

-J nA20 msec

FI G. 6. Effect of TEA and 4-AP on the ability of 5-HT to modulate membrane currents. Membrane currents wereelicited by voltage-clamp pulse s from -70 to +20 mV. Al: current response s were elicited first in ASW (trace a), followingaddition of 2 mM TEA (trace b) to block IK.CYa, and following the addition of 5-HT (35 PM) to the bath already containingTEA (trace c). A2: 5-HT difference current was isolated by subtracting the current response elicited in the presence of 5-HT(and low TEA) from the current response elicited in the presence of low concen trations of TEA (b - c). This differencecurrent is representative of results obtained in 16 different preparations. BI: in a different cell, current response s wereelicited first in ASW (trace a), following addition of 1.5 mM 4-AP (trace b) to block IK,I., and following the addition of 5-HT(35 pM) to the bath containing 4-AP (trace c). B2: 5-HT difference current was isolated by subtracting the current responseelicited in the presence of 5-HT (and 4-AP) from the current response elicited in the presence of 4-AP (b - c). This differencecurrent is representative of results obtained in 6 different preparations. Thes e results indicate that agents (low TEA) thatblock ZK.ca do not block the voltage-dependent compone nt of I5-HT, whereas, agents (4-AP) that block &I block thevoltage-dependent compon ent of&r. Moreover, when the voltage-dependent compone nt of 1s-HT is blocked by 4-AP, therelatively voltage-independent compon ent of I 5 n-r- is revealed. Thus the pharm acologic sensitivity of the voltage-dependentcompon ent of I 5-Hr appears to be identica l to the pharma cologic sensitivity of IKqI/.


9/15

MODULATION OF K+ CURR ENTS 673tions of 4-AP, whereas IKCa is not (see Figs. 3B and 4).Therefore, we examined the sensitivity of the voltage-de-pendent component of 15-HT o 4-AP by isolating 15-Hr nsolutions that contained 4-AP (Fig. 6B). Membrane cur-rents were elicited by voltage-clamp pulses o +20 mV; firstin ASW, following addition of 1.5 mM 4-AP and followingaddition of 5-HT to the solution already containing 4-AP(Fig. 6Bl). As shown in Fig. 6B2, 4-AP blocked the volt-age-dependent component of 15-HT also see Fig. 8). The5-HT difference current that was solated in the presence of4-AP revealed only the residual voltage-independent com-ponent that is identical to the S current. [Previous work hasshown that the S current is relatively insensitive to 4-APand has a & for 4-AP of 10 mM (Shuster and Siegelbaum1987; Brezina 1988).] Thus an agent (4-AP) that blocks Ik L(but not JK,Ca) lso blocks the voltage-dependent compo-nent of 15-HT.To more completely compare the sensitivities to TEA ofIk Iwand the voltage-dependent component of &iT, weexamined IsmHTrom sensory neurons that were bathed inASW (Fig. 7A) and from neurons bathed in ASW thatcontained various concentrations of TEA (Fig. 7, B-E). Ineach case, the voltage-clamp pulse was from -70 to +20mV. Thus, in the experiments of Fig. 7, different prepara-tions were first exposed to a specific concentration of TEAand then the ability of 5-HT to modulate the membranecurrents was examined. At concentrations of 2 and 5 mMTEA, the voltage-dependent component of 15-HTwas stillpresent (Fig. 7, B and C). These concentrations of TEA canbe effective in blocking Ik Ca:,but have lessof an effect onIKsl. (Hermann and Go&an 198 1b; Klein et al. 1982;Walsh and Byrne 1989) (also seeFigs. 3, 4, and 9). There-fore, it is unlikely that the complex kinetics of &-nT can beaccounted for by an increase in the amplitude of Zk ca. Thevoltage-dependent component of &i-r was blocked only atthe high concentrations of TEA that are also effective inblocking IK.I. (Fig. 7, D and E; note the change in the valueof the calibration bar). 5-HT difference currents that wereisolated in ASW containing 100 mM TEA reveal only theresidual voltage-independent component that is identicalto the S current obtained at less depolarized membranepotentials (compare Figs. 7E and 2C2).

A ASW

B 2 mM TEA

\ ._

IFigure 8 compares the current-voltage (Z-I/) relation-ships of 5-HT difference currents that were isolated fromsensoy neurons under t hree conditions: 1) from sensoryneurons bathed first in ASW and then in 5-HT, 2) fromsensory neurons first bathed in ASW that contained 100mM TEA (to reduce Zk,caand Ik i) and then in 5-HT, and

3) from sensory neurons bathed first in 2 mM 4-AP (toblock Jk J,)and then 5-HT. In all three cases, he membranecurrents were elicited by voltage-clamp pulses from -70mV to membrane potentials ranging between -40 and +30mV. The amplitudes of 5-HT difference currents weremeasured at the end of the voltage-clamp pulses and wereplotted such that a positive value represented a decreaseand a negative value represented an increase n the outwardmembrane currents. In cells exposed to ASW and then to5-HT, the relatively voltage-independent component of1 -H-r was evident during voltage-clamp pulses to -40 mVthrough ~0 mV (also seeFig. 2C2). The downward shift inthe I-V relationship of &-HT at 0 mV reflects the develop-ment of the voltage-dependent component of &-HT(also see

C 5 mM TEA

D 50 mM TEA

E 100 mM TEA

I 5nA

I 15nA

I 15nA

I 5 nA

I 5 nA20msecFIG. 7. Effects of preexposure to different concentrations of TEA on

the ability of 5-HT to modulate membrane currents. Each trace is a 5-HTdifference current from a different experiment and in each experiment thecell was maintained in either ASW (A) or ASW containing the indicatedconcentration of TEA (B-E). All voltage-clamp pulse s were from -70 to+20 mV. 5-HT difference currents were isolated by subtracting the currentresponse s elicite d following bath application of 5-HT (30 PM) from thecurrent responses elicited prior to the application of 5-HT (see Figs. 1 and64. Between 5 and 15 preparations were examined for each of the con-centrations of TEA indicated, and similar results were obtained in allpreparations (also see Fig. 9). Note chan ges in ordinate scale s. Theseresults indicate that increased concentrations of TEA, which increase theblock of &., block the voltage-dependent compone nt of 15-H*. In highconcen trations of TEA the relatively voltage-independent compone nt ofJS-rjT. s revealed.

Fig. 2Cl). Of particular significance is the observation thatthe concentrations of 4-AP and TEA that block 1k v alsoblock this downward shift in the I- V relationship for &-nT.Since 4-AP does not block 1k ca, the absenceof the down-


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. ASW aA 4-AP (2 mM) Ir#In TEA (IOOmM) ,k

,I.

,,,

Vm (mV) \

-3 -L -1

FIG. 8. Current-voltage relat ions hips for the relatively voltage-inde -pendent and voltage-dependent compo nents of 15-ijl.. Current-voltage(I-I,) relationships are shown for 5-HT difference currents that were iso-lated under 3 conditions : I) from neurons bathed in ASW, 2) fromneurons bathed in ASW conta ining 2 mM 4-AP, and 3) from neuronsbathed in ASW containing 100 mM TEA. Amplitudes of the 5-HT differ-ence currents were measured at the end of the voltage-clamp pulses . Dataare plotted such that a positive value represents a decrease in the outwardmembrane current and a negative value represents an increase in theoutward membrane current. Each point represents the average of between4 and 15 experiments. For each average, the coefficient of variation was nogreater than 7% of the mean value. In ASW, I 5 HT is present at potentials asnegative as -40 mV and increases with depolarization in a relativelyvoltage-independent manner up to a potential of ~0 mV. At potentialsmore positive than 0 mV, the highly voltage-dependent componen t of&-HT is observed. Pretreatment with 2 mM 4-AP does not affect &-nT in therange of -40 to 0 mV but completely blocks the highly voltage-dependentcompone nt of I _HT normally observed at potentials more depolarizedthan 0 mV. T EA ( 100 mM) also blocks the highly voltage-dependentcompone nt of I 5 i1 I and has a slight effect on the relatively voltage-inde-pendent compone nt of &-HT. These results indicate that pharma cologicagents that block I K;.I. also block the voltage-dependent componen t of1-H I *

ward shift in the I- I/relationship for &,- in the presence of4-AP indicates again that modulation of Ik ca cannot ac-count for the complex kinetics and voltage dependence of15-HT. It is also interesting to note that this concentration of4-AP had absolutely no effect on the component of &iTelicited by voltage-clamp pulses to membrane potentialsmore negat ive than 0 mV. In contrast, high concentrationsof TEA reduced the amplitude of 15-HT elicited at thesesame membrane potentials. Therefore, it appears that therelatively voltage-independent component of 15-HT isslight ly sensitive to TEA and insensitive to 4-AP. Thispharmacologic sensitivity of the relatively voltage-indepen-dent component of I 5-H-r is identical to that of the S current(Klein et al. 1982; Brezina et al. 1987, 1988; Shuster andSiegelbaum 1987; Walsh and Byrne 1989).Figure 9 illustrates the dose-response relationships forthe effects of TEA on an Zk ca-like current, IK v, and thetwo components of 15-ljT. The relatively voltage-indepen-dent component of 1_ H-i- was isolated from voltage-clamppulses to -20 mV (I 5-HT,-20), which did not activate thevoltage-dependent component (e.g., Fig. 1). The voltage-

Log D-m (M)FIG. 9. TEA dose-response relations for IK,


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MODULATION OF K- CURRENTS 675

see Stanfield 1983; Hille 1984; Rudy 1988) indicate thateach K+ channel is blocked by a single molecule of TEA.Thus the block of KS currents by TEA can be fit by aone-to-one binding reaction, which assumes that the per-centage of K+ current remaining is equal to the number ofKS channels not occupied by a molecule of TEA: % con-trol = 100 - 100X ([TEA]/& + [TEA])), where I& is theapparent dissociation constant. The Ik c,-like current wasthe most sensitive to TEA with an estimated I& of 0.4 mM.Ik LF nd &-r +70 had very similar sensitivities to TEA withestimated Kd of 8 and 5 mM, respectively. Therefore, itappears that I _HT,+zo represents the serotonergic modula-tion of a membrane current that has properties closely re-sembling Ik, I ,. Finally, 15-H-r -20 had an estimated I& of ~92mM. These values for Kd are close to those previously re-ported by others (Thompson 1977; Hermann and Gorman198 1b; Brezina et al. 1987, 1988; Shuster and Siegelbaum1987).Modhtion of membrane currents by CAMP

Previous work on sensory neurons has shown that sero-tonergic modulation of the S current is mediated via cyclicAMP (CAMP) (Siegelbaum et al. 1982, 1987; Camardo etal. 1983; Walsh and Byrne 1984, 1989; Ocorr and Byrne1985; Pollock et al. 1985; Shuster et al. 1985; Belardettiand Siegelbaum 1988). We were interested, therefore, indetermining whether CAMP also mediates the serotonergicmodulation of Ik TY.f CAMP does mediate both actions of5-HT, then the application of a membrane-permeable ana-logue of CAMP should produce the same modulation ofmembrane currents as 5-HT (e.g., Fig. l), and preexposureto a saturating concentration of CAMP should occlude anyfurther modulat ion of membrane currents by 5-HT. Thuswe compared the modulatory actions of 8-pcpt-CAMP and5-HT on membrane currents (Fig. 10). Membrane currentswere elic ited by voltage-clamp pulses to +20 mV; first inASW, following addition of 8-pcpt-CAMP (50 PM) andfollowing the addition of 5-HT (30 PM) to the bath alreadycontaining 8-pcpt-CAMP (Fig. IOA). Applicat ion of 8-pcpt-CAMP reduced the total membrane current at the endof the voltage-clamp pulse but had little effect on totalmembrane current early during the voltage-clamp pulse.The membrane current that was modulated by 8-pcpt-CAMP was isolated by subtracting the current responseelic ited in the presence of 8-pcpt-CAMP from the currentresponse elic ited in ASW (Fig. lo@. The resulting CAMPdifference current (IcAMP) was similar to the relatively volt-age-independent component of &-HT that can be isolated inASW at hyperpolarized membrane potentials (e.g., Fig.2U), and at depolarized membrane potentials when Ik L/has been blocked by 4-AP or high concentrations of TEA(e.g., Figs. 682 and 7E). Increasing the bath concentrationof 8-pcpt-CAMP by an order of magnitude did not produceany further modulation of membrane currents, and thepreexposure to 50 PM of 8-pcpt-CAMP occluded additionalmodulation of the relatively voltage-independent compo-nent of 15-H-r by subsequent applicat ion of 5-HT (Baxterand Byrne 1987). Thus 50 PM 8-pcpt-CAMP appeared toproduce a maximal effect on membrane currents and ap-peared to modulate only the relatively voltage-independentcomponent of 15-HT.

A Step to +20 mV

b + CAMP

c (ASW + CAMP) + 5HT

B Difference Currents

I 0 nAI20 msec

FIG. 10. Comparison of the modulatory effects of CAMP and 5-HT onmembrane currents. A: membrane currents were elicited by voltage-clamppulse s from -70 to +20 mV in ASW (trace a), following the addition of8-pcpt-CAMP (50 PM) (trace b), and following the addition of 5-HT (30PM) to the bath already containing 8-pcpt-CAMP (trace c). B: CAMPdifference current (&*& was isolated by subtracting the current resp onseelicited in the presence o f 8-pcpt-CAMP from the current response elicitedin ASW (a - b). The 5-HT difference current (Js+,.J was isolated bysubtracting the current response elicited the presence of 5-HT (and 8-pcpt-CAMP) from the current respo nse e licited in ASW containing 8-pcpt-CAMP (b - c). This difference current is representative of resultsobtained in 9 different preparations. Thes e results indicate that CAMPmediates the serotonergic modulation of the S current, but does not medi-ate the modu lation of a voltage-dependent compon ent.

Although 8-pcpt-CAMP mimicked the actions of 5-HTon the S current, 8-pcpt-CAMP failed to modulate Ik V.The addition of 5-HT to the bath, which still contains the8-pcpt-CAMP, produced additional modulation of mem-brane currents (Fig. IOA, trace c). Application of 5-HTreduced the membrane current early during the voltage-clamp pulse and increased the membrane current at theend of the voltage-clamp pulse. The modulatory effect of5-HT was isolated by subtracting the current responseelic-ited in the presence of 5-HT (plus 8-pcpt-CAMP) from the


12/15

676 D. A. BAXTE R AND J. H. BYRNEcurrent elicited in ASW and 8-pcpt-CAMP (Fig. 1OB). ThisI 5-nT waveform displayed the characteristic voltage-depen-dent component. These results indicate that the modula-tion of IK L by 5-HT is not mediated by elevated levels ofCAMP. Furthermore, these results further support the find-ing that there are at least two distinct components to 15-H-r.Modulation ofmembrane currents by SCPh

To provide further support for the suggestion that themodulation of I K,I, by 5-HT is not mediated via CAMP, weA Step to +20 mV

B Difference Currentsn 1%HT (b - c)

20 r .,,,.IA /I 1 SCPb///

a ASWb +scPt,

\J 20 nA20 msec

A 1CAMP

) + 5-HT

(a b)

I 5 nA-I 20 msec

FIG. 11. Comparison of the modulatory effects of SCPh and 5-HT onmembrane currents. A: membrane currents were elicited by voltage-clamppulses from -70 to +20 mV in ASW (trace a), following the addition ofSCPb (20 PM) (trace b), and following the addition of 5-HT (20 PM) to thebath already containing SCP,., (trace c). B: SCPb difference current (Iscpr,)was isolated by subtracting the current response elicited in the presence ofSCP,, from the current response elicited in ASW (a - b). This differencecurrent is representative of results obtained in 4 different preparations.The 5-HT difference current was isolated by subtracting the current re-sponse elicited in the presence of 5-HT (and SCPhj from the currentresponse elicited in ASW containing SCPb (b - c). These re sults indicatethat SCPb, mim ics the actions of 5-HT on the S current, but does notmodulate an additional voltage-dependent compone nt as does 5-HT.

? I5-HTFIG. 12. The current-voltage (Z-V) relationships are shown for CAMP

and SCPh difference currents that were isolated from neurons bathed inASW. [The data for 5-HT d ifference currents isolated in ASW (Fig. 8) arereplotted in this figure for the purpose of compa rison.] The amplitudes ofthe CAMP and SCPh difference currents were measured at the end of thevoltage-clamp pulses , and the data are plotted such that positive valuesrepresent a decrease in the outward membrane current (e.g., see Figs. 10and 11). Each p oint represents the average of between 4 and 15 experi-ments. For each average, the coefficient of variation was no greater than13% of the mean value.

compared the modulatory effects of small cardioactivepeptide b (SCPb) and 5-HT. In sensory neurons, the actionsof SCPb parallel the actions of 5-HT by elevating intracel-lular levels of CAMP, which in turn results in the closure ofthe S channel (Abrams et al. 1984; Ocorr and Byrne 1985,1986). Thus we compared the modulatory actions of SCPband 5-HT on membrane currents. Membrane currentswere elicited by voltage-clamp pulses to +20 mV first inASW, following addition of SCPb (20 PM) and followingthe addition of 5-HT (20 PM) to the bath already contain-ing SCPb (Fig. 1 A, trace c). The action of SCPb paralleledthat of CAMP and reduced the total membrane current atthe end of the voltage-clamp pulse while having little effecton total membrane current early during the voltage-clamppulse. The application of 5-HT to the bath, which stillcontained the SCPb, produced additional modulation ofmembrane currents (Fig. 1 IA). 5-HT reduced the mem-brane current early during the voltage-clamp pulse andincreased the membrane current at the end of the pulse.The membrane current that was modulated by SCPb wasisolated by subtracting the current response elicited in thepresence of SCPb from the current response elicited inASW (Fig. 1lB). The resulting SCPb difference current(lsCPb) was similar to JcAMPand the relatively voltage-inde-pendent component of &-HT (Figs. 2C2 and 1OB). The mo-dulatory effect of 5-HT was isolated by subtracting thecurrent response elicited in the presence of 5-HT (plusSCPb) from the current elicited in ASW and SCPb (Fig.1OB). This &-H-r waveform displayed the characteristic volt-age-dependent component.The differential effects of 5-HT, CAMP, and SCPb onmembrane currents are illustrated by the Z-V relationshipsof LAMP $ km 9and 15-n-r Fig. 12). As described above (Fig.8) in ASW, the downward shift in the I- I relationship of&-nT at membrane potentials above 0 mV indicates thedevelopment of the voltage-dependent component of &-HT.


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MODULATION OF K+ CURRENTS 677The observation that the 1-V relationships for IcAMP andlscPb for membrane potentials above 0 mV do not show thedownward shift (Fig. 12) further supports the finding thatthere are two distinct components to &-HT, only one ofwhich is modulated via elevated levels of CAMP.DISCUSSION

We conclude that in somata of sensory neurons isolatedfrom pleural ganglia 5-HT modulates at least two KS cur-rents that are elicited by brief voltage-clamp pulses. Onecomponent has properties consistent with the previouslydescribed S current (Klein et al. 1980, 1982; Siegelbaum etal. 1982, 1987; Camardo et al. 1983; Walsh and Byrne1984, 1989; Ocorr and Byrne 1985; Pollock et al. 1985,1987; Scholz and Byrne 1987, 1988). It is relatively voltageindependent, activates relatively slowly, does not inacti-vate, is not blocked by 2 mM 4-AP, is relatively insensitiveto TEA, and under proper pharmacologic conditions canbe isolated and observed at membrane potentials rangingbetween -40 and +30 mV. The other component hasproperties similar to Ik I. It is voltage dependent withcomplex kinetics, and it is blocked by 4-AP and TEA.Therefore, we propose that in addition to the S current,5-HT also modulates Ik cw , ossibly by slowing its activa-tion and inactivation kinetics.There are several possible reasons why previous investi-gations have not observed the voltage-dependent compo-nent of &.I in the sensory neurons of Aplysia. First, thiscomponent, like 1k, hs, s susceptible to cumulative inacti-vation during repetitive depolarizations. For example, dur-ing voltage-clamp pulses to +20 mV that were separated by90 s and preceded by hyperpolarizations, we observed out-ward membrane currents that reached an average peakamplitude of 148 nA within 20 ms (e.g., Fig. 2A). Eliminat-ing the hyperpolarizing prepulse and shortening the inter-pulse interval to 10 s reduced the outward current by anorder of magnitude and significantly altered the actions of5-HT on these attenuated membrane currents (data notshown). In addition, the cumulative inactivation is temper-ature sensitive, and inactivation is more pronounced atroom temperature (22 to 2sC) than at the normal physi-ological temperature of 1SC used in this study (data notshown). Second, 5-HT often does not significantly reducethe maximum conductance of this voltage-dependent com-ponent (e.g., Figs. 1, 2, 6, and 11). Therefore, examiningthe effects of 5-HT on the peak amplitudes of membranecurrents would not detect the changes in the kinetics thatwere revealed by difference currents. Finally, the possibilitythat abdominal and pleural sensory neurons respond dif-ferently to 5-HT cannot yet be ruled out.Previous patch-clamp studies have shown that in ab-dominal sensory neurons 5-HT closes the S channel by aCAMP-dependent phosphorylation of a protein closely as-sociated with this K channel (Siegelbaum et al. 1982;Shuster et al. 1985). Of the two currents modulated by5-HT that are described in this paper, only the relativelyvoltage-independent component appears to be sensitive toelevated levels of intracellular CAMP (see also Walsh andByrne 1984, 1989; Pollock et al. 1985). Bath applications ofCAMP analogues mimic the action of 5-HT on the rela-tivelv voltage-independent component of IqmFIT. but do not

mimic the action of 5-HT on the voltage-dependent com-ponent of I 5-n-r (Baxter and Byrne 1987). Furthermore,SCPb, which elevates levels of CAMP, modulates only therelatively voltage-independent component of 15-n-r. Theseresults indicate not only that the modulation of JK v by5-HT may require another second messenger system: butalso provide additional evidence that there are two separatecurrents modulated by 5-HT. Although the mechanism bywhich 5-HT modulates IK,C is not known, a tentative hy-pothesis can be advanced. We have observed that the rela-tionship between the time-to-peak for Ik v and membranevoltage is shifted to more positive membrane potentials by5-HT (data not shown). Recent experiments in squid axonhave indicated that protein phosphorylation can result in ashift in the voltage dependence of both the activation andinactivation of I k,I/ (Bezanilla et al. 1986; see also Lagruttaet al. 1989). A similar phosphorylation could account forthe modulation of 1k h in the sensory neurons.While it is not yet clear whether both components of15-nT are modulated during endogenous nervous activity, itis interesting to note that the somata of these sensoryneurons are enveloped by serotonergic varicosities (Lo etal. 1987; Zhang et al. 1988). Physiological stimuli thatcause release of 5-HT from these varicosities would haveprofound effects on the electrophysiological activity of thesensory neurons. Since the relatively voltage-independentcomponent (S current) does not inactivate and is a promi-nent current at the resting membrane potential, it reducesthe excitability of sensory neurons by shunting depolariz-ing currents. Inhibiting the S channel by bath applicationof CAMP analogues doubles the number of action poten-tials stimulated during 1 s depolarizing current pulses, andmodestly broadens the action potential (Klein et al. 1986;Baxter and Byrne 1987). Although & is not activated atthe resting membrane potential, it increases dramaticallywith large depolarizations, becoming the predominant out-ward current. Thus activation of IKsl. contributes signifi-cantly to the repolarization phase of the action potential.The modulation of I k,v by 5-HT causes a significant de-crease in this outward current and a threefold increase inspike duration (Baxter and Byrne 1987 and manuscript inpreparation). Thus closure of S channels may account pri-marily for the enhanced excitability (Klein et al. 1986;Baxter and Byrne 1987), and modulation of Ik crmay ac-count primarily for the broadening of the action potentialthat is observed in the pleural sensory neurons during ap-plication of 5-HT.The pharmacologic methods and the parameters for thevoltage-clamp protocol that were used in this study do notrule out the possibility that other currents are modulatedby 5-HT. Indeed, Walsh and Byrne (1989) found that 5-HTreduces a Ca2+-sensitive K+ current that is blocked by lowconcentrations of TEA but that does not appear to be acti-vated by brief voltage-clamp pulses. Although a possibilityexists for a TEA-insensitive species of lkCa in these cells(e.g., Hermann and Hartung 1983; Deitmer and Eckert1985; Kehoe 1985), it is unlikely that the modulation by5-HT of a TEA-insensitive 1k Cacould account for the volt-age-dependent component of 15-uT. To account for theblock of the downward shift in the I- V relationship of IS-n1by 4-AP (Fig. 6), such a current would have to be alsosensitive to 4-AP. At present. none of the Ca2+-activated


14/15


K currents so far described have been shown to be blockedby aminopyridines (Thompson 1977; Adams et al. 19SOb;Hermann et al. 198 la, 1983; Stanfield 1983; Hille 1984;Latorre et al. 1984; Thomas 1984; Deitmer and Eckert1985; Kehoe 1985; Mallart 1985; Blatz and Magleby 1987;Ritchie 1987; Smart 1987; Cook 1988; Rudy 1988). Fur-thermore, it is unlikely that the kinetics of IKca could ac-count for both the initial upward peak and the late down-ward shift in the voltage-dependent component of 15-HT(e.g., Fig. lB2). There also remains the possibility that5-HT may modulate inward currents that are not affectedby 4-AP or TEA (e.g., Hockberger and Connor 1984; Pell-mar 1984; Paupardin-Tritsch et al. 1986; Edmonds et al.1987; Tsien 1987; Braha et al. 1988).The results presented in this paper and by others indicatethat the modulatory effects of 5-HT on the cellular proper-ties of sensory neurons in Aplvsia involve several compo-nents. At least three different I? currents are altered: the Scurrent (Klein et al. 1980, 1982; Pollock et al. 1985), aslowly activating Ca2+ -sensitive K current (Walsh andByrne 1989) and IK.IIV.There is also an increase in the levelsof free intracellular Ca* following application of 5-HTthat is not merely secondary to the changes in Kf currents(Boyle et al. 1984). In addition, 5-HT has other actions;including, translocation of the phospholipid-dependent Ckinase (Hochner et al. 1986a; Sacktor et al. 1986), stimula-tion of protein synthesis that is required for the develop-

BAXTER, D. A. AND BYRNE, J. H. Reduction of voltage-activated IS+currents by forskolin is not mediated by CAMP in pleural sensoryneurons of Aplysia. Sot. Neurosci. Abstr. 14: 153, 1988.

BELARDETTI, F. AND SIEGELBAUM, S. Up- and down-modulation of singleKS channel function by distinct second m essengers. Trends Neurosci.11: 232-238, 1988.

BEZANILLA, F., CAPUTO, C., DIPOLO, R., AND ROJAS, H. Potassium con-ductance of squid giant axon is modulated by ATP. Proc. Natl. Acad.Sci. USA 83: 2743-2745, 1986.

BLATZ, A. L. AND MAGLEBY, K. L. Calcium-activated potassium chan-nels. Trends Neurosci. 10: 463-467, 1987.BOYLE, M. B., KLEIN, M., SMITH, S. J., AND KANDEL, E. R. Serotoninincrease s intracellular Ca transients in voltage-clamped sensoryneurons of Aplysia californica. Proc. Natl. Acad. Sci. USA 8 1:7642-7646, 1984.

BRAHA, L., KLEIN, M., AND KANDEL, E. R. Phorbol ester increase s Ca*current in Aplysia sensory neurons. Sot. Neurosci. Abstr. 14: 644, 1988.

BREZINA, V. Guanosine S-triphosphate analogue activates potassiumcurrent modulated by neurotransmitters in Aplysia neurones. J. Phys-iol. Lond. 407: 15-40, 1988.

BREZINA, V., ECKERT, R., AND ERXLEBEN, C. Modulation of potassiumcondu ctances by an endogeno us neuropeptide in neurones of Aplysiacaltfornica. J. Physiol. Lond. 382: 267-290, 1987.

BYRNE, J. H. Analysis of ionic conductance mechanisms in motor ce l lsmediating inking behavior in Aplysia cahfornica. J . Neurophysiol. 43:630-650, 1980.

BYRNE, J. H. Neural and molecular mech anisms underlying informationstorage in Aplysia: implicatio n for learning and memory. Trends Neu-rosci. 8: 478-482, 1985.BYRNE, J. H. Cellular analysis of assoc iative learning. Physiol. Rev. 67:

329-439, 1987.

Aplysia by serotonin and CAMP-dependent protein phosphorylation.Cold Spring Harbor Symp. Quant. Biol. 48: 2 13-220, 1983.CAREW, T. J. Cellular and molecular advances in the study of Aplysia. In:The Neural and Molecular Bases qf Learning: Dahlem Konferenzen,

edited by J.-P. Changeux and M. Konis hi. New York: Wiley, 1987, p.

CAMARDO, J. S., SHUSTER, M. J., SIEGELBAUM, S. A., AND KANDEL, E. R.Modulation of a spe cific potassium chann el in sensory neurons ofment of long-term presynaptic facilitation and increasedexcitability (Montarolo et al. 1986; Dale et al. 1987) andmobilization of releasableneurotransmitter (Gingrich et al.1985, 1987, 1988; Hochner et al. 1986a). Thus there aremultiple sites for plasticity within a single neuron, and 177-203.while complex, all of the effects of 5-HT may act synergis- CAREW, T. J. AND SHALEY, C. L. Invertebrate learn ing: from behavior totically to facilitate synaptic transmission at the sensory to molecules. Annu. Rev . Neurosci. 9: 435-487, 1986.motor neuron synapse. CONNOR, J. A. AND STEVENS, C. F. Voltage clamp studies of a transientoutward current in gastropod neural somata. J. Physiol. Lond. 213:We thank Drs. L. Cleary, S. Critz, E. Kande l, M. Klein, and K. Scholz 21-30, 1971.

for their h elpful comm ents on an earlier draft of this manu script. COOK, N. S. The pharmacology of potassium chann els and their therapeu-This research was sponsored by the Air Force Office of Scien tific Re- tic potential. Trends Pharmacol. Sci. 9: 2 -28, 1988.

search, Air Force Command, USAF, under Grants AFOSR 87-0274 and DALE, N., KANDEL, E. R., AND SCHACHER, S. Serotonin produces long-National Institute of Mental Health Award K02 MH-00649. term change s in the excitability of Aplysia sensory neurons in culture

Address for reprint requests: D. A. Baxter, Dept. of Neurobiology and that depend on new protein synthesis. J. Neurosci. 7: 2232-2238, 1987.Anatomy, The University of Texas Medical Schoo l, P.O. Box 20708, DEITMER, J. W. AND ECKERT, R. Two compon ents of Ca-dependentHouston. TX 77225. potassium current in identified neurones of Aplysia californica.Pfluegers Arch. 403: 353-359, 1985.Received 24 August 1987: accepted in final form 10 April 1989. EDMONDS, B. W., KLEIN, M., AND KANDEL, E. R. A compone nt ofREFERENCESABRAMS, T. W.. CASWIJNXI, V. F., CAMARDO, J. S., KANDEL, E. R.,

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ADAMS, D. J., SMITH., S. J., AND THOMPSON, S. H. Ionic currents inmolluscan soma. Annu. Rev. Neurosci. 3: 14 l- 167, 1980b.

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BAXWR, D. A. AND BYRNE, J. H. Serotonin-modulated membrane cur-rents Ap/y.sia tail sensory neurons. Sot. Neurosci. Abstr. 12: 765, 1986.

BAXTER, D. A. AND BYRNE, J. H. Modulation of membrane currents andexcitability by serotonin and CAMP in pleural sensory neurons of Aply-sia. Sot. Neurosci. Abstr. 13: 1440, 1987.

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FINKEL, A. S. AND GAGE, P. W. Conventional voltage clamping with twointracellular microelectrodes. In: Voltage and Patch Clamping WithMicroelectrodes, edited by T. G. Smith, Jr., H. Lecar, S. J. Redman, andP. W. Gage. Was hington, DC: Am. Phy siol. Sot., 1985, p. 47-94.GINGRICH, K. J., BAXTER, D. A., AND BYRNE, J. H. Mathematic model o fcellular mech anisms contributing to presynaptic facilitation. Brain Res.B2411. 1: 5 13-520, 1988.

GINGRICH, K. J. AND BYRNE, J. H. Simulation of synaptic depression ,posttetanic potentiation, and presynaptic facilitation of synaptic poten-tials from sensory neurons mediating gill-withdrawal reflex in Aplysia.J. Neurophwysiol. 53: 652-669, 1985.

GINGRICH, K. J. AND BYRNE, J. H. Single-c ell neuronal model for asso-ciative learning. J. Neurophysiol. 57: 1705- 17 15, 1987.

GORMAN, A. L. F. AND THOMAS, M. V. Potassium conductance andinternal calcium accumulation in a molluscan neurone. J. Physiol.Lond. 308: 287-3 13, 1980.

HAWKINS, R. D., CLARK, G. A., AND KANDEL, E. R. Cell b iologica lstudies of learning in simple vertebrate and invertebrate systems. In:Handbook of Physio logy. The Nervous System. Higher Functions of theBrain. Bethesda, MD: Am. Ph ysiol. Sot., 1986, sect. 1, vol. V, chapt. 2,p. 25-83.

HERMANN, A. AND GORMAN, A. L. F. Effects of 4-aminopyridine on


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