Activators of Protein Kinase C Mimic Serotonin ... - Semantic Scholar

JOURNALOF NEUROPHYSIOLWY Vol. 72, No. 3, September 1994.

in C’.S..-t.

Activators of Protein Kinase C Mimic Serotonin-Induced Modulation of a Voltage-Dependent Potassium Current in Pleural Sensory Neurons of ApZ’k SHUZO SUGITA, DOUGLAS A. BAXTER, AND JOHN H. BYRNE Department of Neurobiology and Anatomy, University of Texas iMedical School at Houston, Houston,

SUMMARY

AND

CONCLUSIONS

INTRODUCTION

1. In the pleural mechanoafferent sensory neurons of Aplysia, serotonin (5HT)-induced spike broadening consists of at least two components: a CAMP and protein kinase A (PKA)-dependent, rapidly developing component and a protein kinase C (PKC)-dependent, slowly developing component. Voltage-clamp experiments were conducted to identify currents that are modulated by PKC and thus may contribute to the slowly developing component of 5-HT-induced spike broadening. 2. We compared the effects of phorbol esters, activators of PKC, on membrane currents with those of 5-HT. Bath application of 5-HT had complex modulatory effects on currents elicited by voltage-clamp pulses to potentials >O mV. The kinetics of both activation and inactivation of the membrane currents were slowed by 5-HT. This led to a decrease in an outward current at the beginning of the voltage-clamp pulse and an increase at the end of the pulse. Previous work has shown that these effects represent, in part, the modulation of a large, voltage-dependent K+ current (IKv) by 5-HT. j. Active phorbol esters mimicked some of the actions of 5-HT on membrane currents in that they slowed activation and inactivation kinetics of current responses to voltage-clamp pulses more positive than 0 mV. This led to a decrease in an outward current at the beginning of the pulse and an increase at the end of the pulse. Because inactive phorbols did not mimic the actions of 5-HT, the effects of active phorbol esters appeared to be PKC specific. In addition, preexposure of the sensory neurons to active phorbol esters appeared to occlude the modulatory actions of 5-HT on I Thus it is likely that modulation of lkv, by 5-HT is mediated, a?case in part, by PKC. 4. To further characterize which currents were modulated by PKC, low concentrations of tetraethylammonium (TEA, 2 mM) were used to block Ca2+-activated K+ current (Irc,&. Low TEA partially blocked the phorbol ester-induced increase of the outward current at the end of voltage-clamp pulses. These results agreed with previous reports that activation of PKC enhanced a fast component of IK,ca in these sensory neurons. Such an enhancement would lead to an increase in outward current that should be blocked by low TEA. Low TEA, however, did not affect phorbol ester-induced decrease of the outward current at the beginning of pulse, where the predominant current is IKv, which is less sensitive to TEA. Nor did low TEA block phorbol ester-induced spike broadening. Thus modulation of Ikv appears to contribute to the PKC-mediated component of 5-HT-induced spike broadening. 5. On the basis of our results and those of others, a scheme is proposed in which the modulatory actions of 5-HT are mediated via two protein kinase systems (the PKC and PKA pathways) that converge and diverge to affect various ionic conductances.

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Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system in which to study the cellular and molecular mechanisms of simple forms of learning such as sensitization (Byrne 1987; Carew and Sahley 1986; Kandel and Schwartz 1982). Sensitization is mediated, at least in part, by the transmitter serotonin (5.HT) and expressed in several forms including facilitation of sensorimotor connections. Serotonin-induced spike broadening is believed to be a key mechanism underlying facilitation of undepressed synapses (Gingrich et al. 1988; Hochner et al. 1986; Kandel and Schwartz 1982). Previously, this broadening was believed to be dependent primarily on CAMP-mediated reduction of a noninactivating, relatively voltage-independent K+ current termed the S-K+ current (1k s) (Kandel and Schwartz 1982; Klein et al. 1982). However, recent evidence suggests that 5-HT-induced somatic spike broadening is composed of, at least, two components: a CAMP-dependent, rapidly developing component of broadening and a CAMP-independent, slowly developing component of broadening (Baxter and Byrne 1990a; Hochner and Kandel 1992; Mercer et al. 199 1; Sugita et al. 1992b). For example, application of CAMP analogues, such as bath-applied 8bromo-CAMP and pcpt-CAMP, or intracellularly diffused Sp-CAMP, can mimic aspects of the rapidly developing component of 5-HT-induced broadening (within 3 min after 5-HT application), but do not mimic the slowly developing component (after 3 min) (Baxter and Byrne 1990a; Goldsmith and Abrams 1992; Hochner and Kandel 1992). In addition, CAMP antagonist, Rp-CAMP, and a peptide inhibitor of PKA inhibit 175% of the rapidly developing component of broadening (Goldsmith and Abrams 1992; Hochner and Kandel 1992), but the inhibition of a slowly developing component by these inhibitors has not been demonstrated. Based on the above considerations, other second messenger systems appear to be involved in the slowly developing component. One possibility is that the diacylglycerol/protein kinase C (PKC) second messenger system mediates some of the actions of 5-HT. Biochemical evidence suggests that 5-HT activates PKC as well as protein kinase A (PKA) (Bacskai et al., 1993; Bernier et al. 1982; Greenberg et al. 1987; Jarrard et al. 1993; Ocorr and Byrne 1985; Pollock et al. 1985; Sacktor and Schwartz 1990; Sossin and Schwartz 1992). We have shown that phorbol esters, activators of PKC, can mimic

0022-3077/94 $3.00 Copyright 0 1994 The American Physiological Society

Al.

.tt I

MODULATORY

AZ. 5HT

Step to +20 mV

EFFECTS OF 5-HT AND PKC

Difference

Current

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(a - b)

a. ASW

B1. Step

20 nA

to o mV

-

Cl.

/IOnA

dd

(5nA

C2

Step to -20 mV

FIG. 1. Modulation of membrane currents by serotonin (5HT). Membrane currents were elicited by voltage-clamp pulses from -70 to -20, 0, and 20 mV in artificial seawater (ASW, a) and after bath application of 5-HT (10 PM, b). Each panel represents averaged data obtained from 6 sensory neurons. The 5-HT difference currents were obtained by subtracting the current responses elicited in 5-HT from those elicited in ASW (i.e., a - b). At 20 mV, 5-HT slowed both the activation and inactivation kinetics of the membrane current. At -20 mV, 5-HT appeared to reduce an outward current with properties similar to Ircs.

a. ASW

MI

1 nA

b. +5-HT

30 ms

the CAMP-independent component of 5-HT-induced broadening (Sugita et al. 1992b). In addition, the PKC inhibitor staurosporine had little effect on the rapidly developing component of 5-HT-induced broadening but did inhibit significantly the slowly developing component. Taken together, these results suggest that 5-HT-induced spike broadening is composed of a cAMP/PKA-dependent, rapidly developing component and a PKC-dependent, slowly developing component. The currents responsible for the slowly developing component of broadening are not known, however. One possibility is that the slowly developing component of broadening is dependent on PIE-mediated modulation of a large, voltage-dependent K+ current &v). Baxter and Byrne ( 1989) found that 5-HT modulates IK,v as well as IK,s. This modulation is complex and due to a reduction in the magnitude of IKv as well as a slowing of both activation and inactivation kinetics of 1&v (White et al. 1992, 1994). In addition, this modulation did not appear to be mimicked by CAMP analogues (Baxter and Byrne 1989, 1990ab). In the present study, we have examined the effects of phorbol esters on membrane currents. We show that the activation of PKC mimics and partially occludes 5-HT-induced modulation of membrane currents at potentials >O mV, where IKv is significantly activated. The results support the hypothesis that PKC modulates Ikv and that this modulation contributes to the slowly developing component of 5=HTinduced broadening.

30 ms

On the basis of our results as well as those of Hochner and Kandel (1992) and Abrams and Goldsmith (1992), a new scheme for 5-HT-induced somatic spike broadening is proposed in which the modulatory effects of 5-HT are mediated via two second messenger systems converging and diverging on multiple ionic conductances. A preliminary report of some of these results was presented in abstract form (Sugita et al. 1992a). METHODS

Measurements

of membrane

current

Isolatedclustersof sensoryneuron somatafrom pleuralganglia were pinned to the floor of a recordingchambercontaining artificial seawater(ASW, Instant Ocean) with 10 mM tris(hydroxymethyl)aminomethane-HCl (pH 7.6), and the preparation was maintained at 15 t_ 1“C (mean t_ SD). Only a singleneuron per cluster wasusedfor any experiment, and, unlessotherwiseindicated,the clusterreceivedonly a singleapplicationof a given agent or concentration of an agent. The sensoryneuronswere voltage clampedat their restingpotentialsand conventionaltwo-electrode voltage-clamp techniques were used to measure membrane currents. As in previous studies(Baxter and Byrne 1989, 1990a; Goldsmith and Abrams 1992;Hochner and Kandel 1992),membranecurrentswereactivated by 200-msvoltage-clamppulsesthat were precededby a 15-s,hyperpolarizing prepulseto -70 mV. This prepulseservesto remove inactivation of membranecurrents and thusallowsfor a morecompleteexamination of the characteristicsand modulation of currents.Adherenceto this protocol also

1242

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S. SUGITA,

D. A. BAXTER,

AND J. H. BYRNE

AZ. PDAc Difference Current

Step to +20 mV

(a - b)

a. ASW 20 nA

40 nA

B1. Step

to o mV

IOnA

5 nA

c

Cl. Step

FIG. 2. Serotonin-induced modulation of membrane currents elicited at potentials more positive than 0 mV were mimicked by 4@-phorbol 12,13-diacetate (PDAc). The procedures were the same as those used for 5-HT (e.g., Fig. 1). Traces are averaged data recorded from 9 sensory neurons except for -20 mV. Trace at -20 mV represents averaged data from 8 sensory neuron, because 1 cell was examined for its response at -30 mV. Like 5-HT, PDAc (3 PM) appeared to slow both the activation and inactivation kinetics of membrane currents elicited at potentials >O mV. PDAc also appeared to affect membrane currents elicited at -20 mV. This effect, however, may not be protein kinase C (PKC)-specific, because inactive cu-phorbol had similar effects (see Fig. 4).

C2

to -20 mV

b. +PDAc 5 nA a. ASW

P

30 ms

makespossiblecomparisonsbetweenthe presentresultsand previous work. At the end of the 200-msdepolarization, the membranepotential wassteppedbackto a value of -70 mV for 500ms. In mostcells,the seriesof three voltage-clamppulsesto membrane potentialsof -20 (or -30 ), 0, and 20 mV wereused.To avoid the cumulative inactivation of membraneconductances,the voltageclamppulseswere separatedby 90 s (seeBaxter and Byrne 1989). Betweenthe voltage-clamppulses,the cell wasreturned to its resting potential. Two stablemembranecurrent responses wereobtained at each voltagein the seriesof voltage-clamppulsesbeforeand after addition of agentsto the bathing solution. Thesetwo responses were averagedoff-line to obtain a singletrace for each potential in the seriesof clamp pulses.The effectson membranecurrents of the agentsaddedto the bath wererevealedby computer subtractionof membranecurrentselicited after drug treatment from membrane currentselicited before treatment.

/InA 30 ms

action potential wasmeasuredasthe time betweenthe peakof the spike and the point of the repolarizing phaseat which the membranepotential was 10%of the peakamplitudeof the spike(Sugita et al. 1992b). After three stable trials, concentrated aliquots of agentswereapplieddirectly to the staticbath. In eachpreparation, data were normalized to the mean of the duration of the three baselinespikesbefore the application of agents.

Chemicals

Two active types of hydrophilic phorbol esters,4P-phorbol 12,13-diacetate(PDAc, Sigma)and 4p- 12-deoxyphorbol 13-isobutyrate (DPB, LC Services),were used. As a control, inactive 4cw-phorbols (Sigma)alsowere used.Stock solutionsof all phorbols ( 10 mM) weredissolvedin dimethyl sulfoxide (DMSO) and storedat -2OOC.The final concentration in the bath was3 PM for PDAc and 4cu-phorboland 2 PM for DPB. The final concentration of DMSO usedto dissolvethe phorbolsdid not exceed0.03% in the bath (v/v). The concentrations of phorbol estersusedin the Measurements of spike duration presentstudy werethe sameasthoseusedpreviously (Sugitaet al. Two-electrodecurrent-clamp techniqueswereused.The mem- 1992b).Previouswork (Critz and Byrne 1992)examinedthe dosebranepotential of the sensoryneuronswasadjustedto -45 mV by responsiverelationshipbetweenconcentrationsof phorbol esters manually injecting the current -30 s beforethe measurementof and the modulation of 1K,caand found that concentrationsof - 1 spikeduration. Individual action potentialswereelicited by pass- PM or higher are necessaryto ensuresaturation of phorbol-meing 3-ms, 5- to 6-nA current pulsesthrough the current passing diated effects. In addition, Goldsmith and Abrams (1992) reelectrodeat an interstimulusinterval of 3 min. The duration of an ported that 500 nM of 4@-phorbol12,13-dibutyrate(PDBu) did

MODULATORY

Al.


A*. DPB Difference Current

Step to +20 mV

-

FIG. 3. Membrane currents were affected by 4p- 12deoxyphorbol 13-isobutyrate (DPB) in a manner similar to that of PDAc. Procedures were the same as for PDAc (e.g., Fig. 2), except that membrane currents at -30 mV were examined instead of -20 mV (Fig. 30. Traces represent averaged data recorded from 10 sensory neurons except for the currents recorded at -30 mV. Trace at -30 mV represents averaged data from 9 neurons, because 1 cell was examined for its response at -20 mV.

G

B2

Step to o mV

-

Cl . Step

(a - b)

20 nA

40 nA

Bj.

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lIOnA

1

1!5nA

C2

to -30 mV

b. +DPB

$ nr

5 nA

4

1 nA

a. ASW

30 ms

30 ms

not reliably facilitate depressedsynapsesin intact sensorimotor connectionand Sossinet al. ( 1993)reportedthat 200 nM of PDBu wasnot a saturatingconcentrationfor the translocationof PKC in intact neurons.Although lower concentrationsof phorbol esters havebeenshownto modulatesomepropertiesof the sensoryneuronsin culture (Brahaet al. 1990),higherconcentrationsappearto be necessaryin intact gangliaand acutely isolatedsensoryneuron somata.We believed that the concentrations of phorbol esters usedin the presentstudy fully activate PKC-mediatedprocesses. Serotonin creatine sulfate (5HT, Sigma)wasdissolvedin ASW and prepared daily. Tetraethylammonium (TEA, Kodak, 100 mM) wasdissolvedin Hz0 and then diluted in ASW to the final concentrationsof 2 mM. Small aliquots of concentrated agents wereaddeddirectly into the recordingchamber.

(Baxter and Byrne 1989; White et al. 1992, 1994). Statistical analyses indicated that both the decrease near the beginning of the pulse ( 10 ms after the beginning) and the increase near the end of pulse (180 ms after the beginning) were significant (the average amplitude t SE of an outward current at 10 ms: 90.6 t 4.6 nA before 5-HT vs. 73.5 t 2.6 after 5-HT, two-tailed paired t test, t5 = 4.50, P < 0.0 1; at 180 ms: 73.1 t 5.9 nA before 5-HT vs. 8 1.6 t 7.1 nA after 5-HT, t5 = 3.86, P < 0.02). At -20 mV, 5-HT appeared to reduce an outward current, which is similar to Ik s (Fig. 1C, 1 and 2) (Baxter and Byrne 1989; Hochner and Kandel 1992; Klein et al. 1982; Pollock et al. 1985). At 0 mV, combined effects of 5-HT on Ik ,v and Ik ,s are apparent (Fig. lB, I and 2).

RESULTS

Membrane

currents modulated

by 5-HT

We first repeated the experiments of Baxter and Byrne (1989) and confirmed their results. Serotonin creatine sulfate (n = 6) slowed both the activation and inactivation kinetics of the current responses elicited by voltage-clamp pulses to membrane potentials >O mV (Fig. lA, I). This resulted in a decrease in the net outward current at the beginning of the pulse and an increase at the end of the pulse (Fig. lA, 1 and 2). This effect developed slowly and became stable 9- 13.5 min after application of 5-HT. These effects represent, in part, a modulation of IKv9 by 5-HT

Phorbol esters mimic SHT-induced modulation of. membrane currents elicited at potentials more positive than 0 mV Application of PDAc (3 PM, n = 9) appeared to slow both the activation and inactivation kinetics of membrane currents elicited at potentials above 0 mV (Fig. 2A, I). Such effects were observed consistently in each of the nine cells examined. The PDAc difference current was very similar to 5-HT difference current (compare Fig. 2A, 2, with Fig. IA, 2). The effect of PDAc developed slowly and became stable 9- 22.5 min after application. This time course corresponds

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S. SUGITA,

AZ.

Step to +20 mV

D. A. BAXTER,

a-phorbol

AND J. H. BYRNE

Difference

Current

(a - b)

a. ASW

B1. Step

to o mV

*

Cl.

20 nA

40 nA

-

Step to -20 mV

IOnA

5 nA

FIG. 4. Inactive a-phorbols had little effect on membrane currents. Procedures were the same as those for PDAc (e.g., Fig. 2). Traces represent averaged data recorded from 4 sensory neurons. Small a-phorbol difference currents likely represent nonspecific effects of phorbol esters and are not the results of the activation of PK.

C2

b. + a-phorbol 1 nA a. ASW

30 ms

well to the effect of phorbol esters on increasing the duration of action potentials (Sugita et al. 1992b). DPB (2 PM) had very similar effects in 9 of 10 cells examined (Fig. 3). Because inactive cu-phorbol (n = 4) did not mimic the effects of active phorbol esters (Fig. 4A), modulation of the current at 20 mV appeared PKC-specific. Statistical analyses indicated that both PDAc- and DPB-induced effects on outward currents at 20 mV are significant (PDAc: at 10 ms, 100.8 t 7.9 nA before vs. 84.4 t 7.2 nA after, t8 = 13.42, P < 0.001; at 180 ms, 79.7 t 4.4 nA before vs. 92.9 t 4.5 nA after, t8 = 5.81, P < 0.001. DPB: at 10 ms, 113.1 t 4.7 nA before vs. 95.3 t 5.2 nA after, tg = 7.33, P < 0.001; at 180 ms, 93.3 t 6.0 nA vs. 102.0 t 7.5 nA after, tg = 2.58, P < 0.05). Unlike 5-HT, neither PDAc or DPB reduced consistently the outward current at -20 mV (Figs. 2C and 3C). Therefore, phorbol esters do not appear to reduce 1k s (however see below and Fig. 6C’). Instead of reducing the outward current, PDAc and DPB slightly increased outward currents elicited at -20 mV. This effect, however, does not seem to be PKC-specific, because inactive cu-phorbol had similar effects (Fig. 4C). Phorbol esters partially occluded SHT-induced modulation of membrane current at 20 mV Because active phorbols mimic 5-HT-induced modulation of membrane current elicited at 20 mV, we examined the relationship between phorbol ester- and 5-HT-induced modulation of the current (Fig. 5). In these experiments (n

30 ms

= 5), the modulation by PDAc or DPB of the current was similar to that produced by 5-HT (compare Figs. 5B and IA, 2). The subsequent application of 5-HT to the bath, which still contained PDAc or DPB, had some additional effects (Fig. 5, A and B). This 5-HT difference current (trace I 5-H-rin Fig. 5B), however, was significantly different from the 5-HT difference current in the absence of active phorbol esters (Fig. IA, 2). Active phorbol esters completely occluded 5-HT-induced increase in the outward current at the end of the voltage-clamp pulse. Indeed, the 5-HT difference current in the presence of phorbol esters indicates the reduction at the end of the voltage-clamp pulse (i.e., the upward deflection at the end of the Zs-HTtrace in Fig. 5B). This slow component of the 5-HT difference current is likely to represent CAMP-dependent modulation of IKs. In addition, preexposure to active phorbol esters significantly reduced the 5-HT-induced modulation of the membrane current at the beginning of the voltage-clamp pulse (compare the fast upward deflection at the 15-H-rtrace in Fig. 5B with that in Fig. lA, 2; at 10 ms after the beginning of the pulse: 7.3 t 1.6% decrease by 5-HT in the presence of active phorbols vs. 18.4 t 3.1% decrease by 5-HT in ASW, tg = 2.95, P < 0.02). This result suggests that phorbol esters partially occluded 5-HT-induced modulation of IK,v. Thus the modulation of I K,V by phorbol esters and 5-HT appears to share some common mechanisms. Alternatively, this ocelusion might be explained by the phorbol ester-induced inhibition of the actions of 5-HT. Recently, prolonged acti-

MODULATORY

A


Step to +20 mv

B

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Difference Currents

a. ASW

-1 Active Phorbol

(a - b)

IOnA

5. Active phorbol esters (PDAc or DPB) partially occluded 5-HT-induced modulation of the membrane current. A: membrane currents were elicited by voltage-clamp pulses from -70 mV to 20 mV in ASW (a), - 13.5 min after the addition of active phorbol esters (b) and -9 min after the addition of 5-HT to the bath already containing active phorbols (c). Traces represent averaged data from 5 sensory neurons. B: active phorbol ester difference current has characteristics similar to those of 5-HT difference current that is shown in Fig. lA, 1. The 5-HT-difference current in the presence of phorbols was isolated by subtracting the current response elicited in the presence of 5-HT (and phorbols) from the current response elicited in ASW containing phorbols (i.e., b - c).

40 ms

FIG.

vation of PKC by phorbol esters was shown to attenuate the ability of 5-HT to activate a CAMP-dependent pathway via possibly the phosphorylation of 5-HT receptors on the sensory neurons (Sugita et al. 1993). PDAc- or DPB-induced modulation of membrane currents persist in the presence of low- TEA concentrations Although Zk v is the dominant current at 20 mV, other outward currents such as Zk,ca also contribute to the net outward current and in turn are modulated by PKC. Critz and Byrne (1992) reported that active phorbol esters enhanced a fast component of ZkCa in these sensory neurons. Thus phorbol ester-induced increase in the outward current at the end of a pulse to 20 mV could be because of a direct modulation of Zkca (Critz and Byrne 1992) or indirect increase in Zk,ca via the phorbol ester-induced increase of a Ca*’ current (Braha et al. 1993). To examine these possibilities, we used a low concentration of TEA (2 mM), which significantly blocks Zk Cabut has little effect on Zk v (Baxter and Byrne 1989; Critz and Byrne 1992; Walsh and Byrne 1989). Low TEA reduced an outward current at the end of pulse (compare a in Fig. 6A, 1, with a in Fig. 2A, 1). In addition, in the presence of TEA, a late component of phorbol ester-induced increase in the outward current was significantly reduced (compare Fig. 6A, 2, with 2A, 2; at 180 ms after the beginning of the pulse: 3.8 t 2.9% increase in TEA vs. 12.9 t 2.7% increase in ASW, t27 = 2.13, P < 0.05). These results suggest that phorbol ester-induced increase in the outward current is because of the modulation of both Zkv. and a fast component of ZkCa. . In contrast to its effects toward the end of the voltage-clamp pulse, low TEA did not significantly reduce the modulation of the early component ofthe membrane current (Fig. 6A, 2; at 10 ms: 20.5 t 1.8% decrease in TEA vs. 16.3 t 1.2% decrease in ASW, t27 =

1.97). Thus phorbol esters appear to modulate a current with voltage-sensitivity and pharmacological properties similar to Zk v (Baxter and Byrne 1989; White et al. 1992, 1994). ’ To examine the possible effect of PKC-induced increase in the fast component of Zkca on spike duration, we also examined the effects of PDAc on spike duration in the presence of 2 mM TEA. PDAc increased the spike duration by 69 + 11% above baseline (Fig. 7). This PKC-induced spike broadening in 2 mM TEA is more than twice that observed in ASW (compare Fig. 7B and Fig. 1E in Sugita et al. 1994). Thus the PKC-induced increase in Zk Caappears to partially counteract the broadening derived from the modulation of Zkv. These results support our hypothesis that PKCinduced spike broadening is due to the modulation of r,,v. Unexpectedly, we observed that active phorbol esters sometimes reduced an outward current with slow kinetics at -20 mV in the presence of 2 mM TEA (Fig. 6C). In contrast, the reduction of the outward current by PKC was not observed in ASW (Figs. 2C and 3C). This discrepancy might be reconciled by assuming that PKC-induced increase in Zk,Ca counteracted PKC-induced modest reduction of the other outward current, such as Zks in ASW. Thus we do not exclude the possibility that activation of PKC may modestly reduce ZKs. It was recently reported that PKC induced a small increase in excitability of the sensory neurons (Sugita et al. 1992b). DISCUSSION PKC mimics SHT-induced

modulation

of ZK.v

Although previous work indicated that activation of PKC plays a key role in the slowly developing component

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S. SUGITA,

A+

D. A. BAXTER,

AND J. H. BYRNE

AZ. Active

Step to +20 mV

Phorbol Difference Current (a - b)

40 nA

B1. Step

IOnA

to o mV

IOnA

Cl.

C2

Step to -20 mV

1 nA

2 nA

30 ms

30 ms

6. Active phorbol (PDAc or DPB)-induced modulation of membrane currents persisted in the presence of low concentrations of tetraethylammonium (TEA). Procedures were similar to those in Fig. 2 except that a low concentration of TEA (2 mM) was used to examine the contribution of ZKca to phorbol ester-induced modulation of the membrane current. Traces represent averaged data from 5- 10 sensory neurons. Low TEA reduced the outward current at the end of pulse (compare a in A, I, with a in Fig. 2A, I). In addition, in the presence of TEA, a late component of the phorbol difference current was reduced (compare A, 2, with Fig. 2A, 2), confirming previous observations that phorbol esters increase a fast component of Zkca (Critz and Byrne 1992). However, TEA did not appear to affect the modulation of the early component of the membrane current. FIG.

of 5-HT-induced broadening (Sugita et al. 1992b), the particular membrane currents that were modulated by PKC were not identified. The present study suggests that activation of PKC modulates IKv in a similar manner to that of 5-HT. The fact that PKC modulates Ikv and produces spike broadening, together with previous simulation studies (Baxter and Byrne 1990~; Belkin et al. 1992; Byrne et al. 1990; Canavier et al. 199 1), also supports the hypothesis that 5-HT-induced modulation of 1k ,v plays a major role in 5-HT-induced broadening. In addition to the modulation of Ikv, our results confirmed the previous work by Critz and Byrne (1992) in that activation of PKC increases a fast component of &. Walsh and Byrne (1989) suggested that there may be two types of I&G in the sensory neurons. One type has slow

kinetics, contributes to the resting membrane conductance, and is decreased by 5-HT. The second type of lkCa has fast kinetics, is activated by relatively brief voltage-clamp pulses at membrane potentials above - 10 mV and is not decreased by 5-HT. The IK,Ca increased by PKC appears to be the fast type. Identity

of IK 9v

Given the role of 1k v in 5-HT-induced spike broadening, it will be important to identify the channels underlying this current and determine whether the pharmacologically identified current is composed of a single or multiple types of K+ channels. Efforts have been made to identify K+ channels in Aplysia (Pfaffinger et al. 199 1; Quattrocki et al.

MODULATORY

a

b. TEA + PDAc J

I


c 4 a, 5

30mV

f TEA

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cn

I

-6

1

I

I

1

I

I

I

-3

0

3

6

9

12

15

Time (min)

3ms

FIG. 7. PDAc produced spike broadening in the presence of 2 mM TEA. A: 2 mM of TEA broadened the action potentials, as compared with those in ASW (see action potentials in ASW in Figs. 1 and 2 of Sugita et al. 1994). PDAc (3 PM, n = 6) induced additional broadening in the presence of TEA. Action potentials shown are before and 15 min after the application of PDAc. Traces represent averaged data from 5 sensory neurons. B: time course of the changes in spike duration after bath application of PDAc. Arrow indicates the application of the PDAc. Error bars represent means * SE.

1994; Zhao et al. 1992), but K+ channels with properties identical to ZKv have not been identified. One type of K+ channel (AKOZa) isolated from Aplysia appears to induce a current with some similarities to Z&v, however (Pfaffinger et al. 199 1). Both ZKv and ZAKOlashow rather fast activation and inactivation kinetics and are more sensitive to 4-AP than TEA. These two currents also have some characteristics that differ, however. One critical difference is that Z&v

A

Early

shows steeper voltage sensitivity than does ZAKOla(Furukawa et al. 1992; White et al. 1994). In addition, ZAKOla shows less sensitivity to TEA than does Z&v. Despite these differences, these two K+ currents may represent similar subfamilies of K+ channels. Interestingly, ZAKOlais not modulated by analogues of CAMP (Furukawa et al. 1992; Pfaffinger et al. 199 1). ZAKOlais modulated by phorbol esters, however, and the modulation is similar to that of Zk,v in

B

/

Later

CAMP

DAG

FIG. 8. A scheme for time-dependent spike broadening by 5-HT. A: at early times (e.g., 3 min), PKA plays a primary role in 5-HT-induced spike broadening via the closure of IKs and modulation of the activation kinetics of IKv; 5-HT also reduces a slow component of Ircca, but it is unlikely to contribute to spike broadening due to its slow kinetics. Dashed lines indicate relatively weaker interactions between protein kinases and membrane currents than solid lines. B: at later times (e.g., 12 min), PKC plays a dominant role in 5-HT-induced spike broadening via the modulation of I,, (and possibly I&. PKC is . . known to increase a fast component of I Kca (not shown), but rt IS not clear whether 5-HT also modulates this current.

\

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S. SUGITA,

D. A. BAXTER,

that there is a slowing of both activation and inactivation kinetics (Kubo and Furukawa 1992). Kubo and Furukawa (1992) identified a specific amino acid, which when phosphorylated, slows the inactivation kinetics. Although an amino acid whose phosphorylation is involved in the slowing of activation kinetics has not been identified, these results suggest that molecular sites involved in the modulation of activation and inactivation kinetics are independent. Another K+ channel (Aplysia Shab) also induces a current with some similarities to 1k,, (Quattrocki et al. 1994). IAplysiaSh&,iS Similar to Ik v in that it shows rather fast activation and inactivation kinetics, but differs in that it is not sensitive to 4-AP. Although Aplysia Shab has consensus sequences that may be phosphorylated by PKA and PKC, the effects of phosphorylation on the current have not been examined. At present, it is not known whether 1&V is mediated by a distinct type of membrane channel or whether it may represent a heterogeneous assemble of I AKOla and rAplysia Shah* Hypothesis for 5-HT-induced

somatic spike broadening

AND J. H. BYRNE

modulates 1k,, as well as I,,,. Support for this latter possibility comes from the experiments using CAMP antagonist, Rp-CAMP (Hochner and Kandel 1992). This antagonist inhibited 50% of 5-HT-induced modulation of activation kinetics of lkv although it did not affect the modulation of inactivation kinetics. Because PKC activators did not completely occlude the modulation of 1k v by 5-HT, our results also suggest that PKA not only modulates 1k s but also may have an effect on the modulation of the activation kinetics of IK,V* Therefore, PKA appears to be involved in some aspects of the modulation of 1k,, by 5-HT at early times. On the basis of our results as well as those of Hochner and Kandel ( 1992) and Goldsmith and Abrams (1992) we propose a time-dependent hypothesis for 5-HT-induced broadening (Fig. 8). In the rapid component of broadening (3 min after application), PKA plays a major role because of the reduction of & and slowing of the activation kinetics of 1k v. For example, Bacskai et al. (1993) found that the concentrations of CAMP reached a peak in 2 min after the application of 5-HT and that they gradually declined with time even in the continuous presence of 5-HT. In the slow component of broadening, PKC plays a major role because of the additional modulation of lkv and the increase in 1ca; 5-HT also appears to reduce a slow component OfIK,,a via a CAMP pathway (Walsh and Byrne 1989). Because of its slow kinetics, the reduction of the slow component of lk,ca may not contribute to the spike broadening. Although PKC was shown to increase a fast component of lkca (Critz and Byrne 1992) it is not known whether 5-HT also increases this component. Interestingly, recent biochemical work supports the timedependent hypothesis we proposed here. Using 2-D polyacrylamide gel electrophoresis, Homayouni et al. ( 1992) examined the effects of different durations of 5-HT application (2 min, 25 min, or 1.5 h) on the phosphorylation of proteins. Then they compared the results of 5-HT with those of PDAc. They found that different sets of proteins are phosphorylated by different durations of 5-HT. Some proteins affected by a 25 min or 1.5 h exposure to 5-HT are also affected by PDAc, but none of the proteins affected by a 2 min exposure of 5-HT are affected by PDAc. The data clearly indicate that the modulatory actions of 5-HT on pleural sensory neurons are mediated via at least two second messenger systems, the cAMP/PKA and DAG/ PKC systems. Moreover, these data suggest that these two systems operate in different time domains. The CAMP/ PKA system appears to mediate rapidly developing effects of 5-HT; whereas the DAG/PKC system appears to mediate more slowly developing effects of 5-HT. The possibility for cross-talk between these two systems also is suggested (Sugita et al. 1993). Thus neuronal plasticity that may contribute to learning and memory appears to be expressed in several overlapping time domains and transitions between short-term and longer term memory may involve several complex and interactive processes.

Sugita et al. (1992b) reported that 5-HT-induced spike broadening appeared to have two distinct time courses, which suggests that more than one mechanism underlies the broadening (see also Sugita et al. 1994). The multiple time courses also have been observed in other laboratories (Hochner and Kandel 1992; Stark et al. 1992). For example, 5-HT increased spike duration by 15% of baseline after 3-min exposure and by 40% of baseline after 12-min exposure (Sugita et al. 1994). The slowly developing component of 5-HT-induced broadening was mimicked by phorbol esters and blocked by staurosporine (Sugita et al. 1992b). In addition, the slowly developing component was not mimicked by CAMP analogues (Baxter and Byrne 1989, 1990a; Hochner and Kandel 1992). Therefore the slowly developing component appears to be primarily dependent on PKC. The present study indicates that the slow component is due primarily to the modulation of lkv by PKC. Recently, it was also shown that 5-HT increases a nifedipine-sensitive Ca*+ current via the activation of PKC (Braha et al. 1993). Although modulation of this current is relatively small compared with the modulation of &, it is plausible that an increase of Ca*+ current also contributes to the spike broadening. The mechanisms of a rapidly developing component appear more complex. Goldsmith and Abrams (1992) found that a PKA inhibitor blocked 75% of this rapidly developing component, and a PKC inhibitor blocked 25% of this component. Similar results were obtained by Hochner and Kandel(1992) and Sugita et al. (1992b). In addition, intracellularly injected Sp-CAMP or extracellularly applied 8bromo-CAMP produced broadening that is comparable with 5-HT-induced broadening at 3 min (Baxter and Byrne 1989; Hochner and Kandel 1992; Sugita et al. 1994). Therefore a major part of a rapidly developing component appears to be dependent on PKA. The remaining question is We thank Drs. T. Abrams and E. Kandel for comments on an earlier how PKA produces this rapidly developing broadening. draft of this paper. One possibility is that this broadening is due totally to the This work was supported by National Institute of Mental Health Awards reduction of lks1 by PKA. Another possibility is that PKA K05 MH-00649 and ROl NS-19895.

MODULATORY


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