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The Contributions of Protein Kinase a and Protein Kinase C to the Actions of 5-HT on the L-Type Ca *+ Current of the Sensory Neurons in Aplysia

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The Contributions of Protein Kinase a and Protein Kinase C to the Actions of 5-HT on the L-Type Ca *+ Current of the Sensory Neurons in Aplysia The Journal of Neuroscience, May 1993, 13(5): 1939-l 851 The Contributions of Protein Kinase A and Protein Kinase C to the Actions of 5-HT on the L-Type Ca *+ Current of the Sensory Neurons in Aplysia 0. Braha,’ B. Edmondq2 T. Sacktor,3 E. R. Kandel,4 and M. Klein5 ‘Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, 2Department of Pharmacology, University College London, London WClE 6BT, United Kingdom, 3Department of Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203, 4Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, New York, New York 10032, and Xlinical Research Institute of Montreal, Montreal, Quebec H2W lR7, Canada In the sensory neurons of Aplysia, 5-HT acts through CAMP Serotonin (5-HT) facilitates the synaptic connection between to reduce current flow through two classes of K+ channels, sensoryand motor neurons of the gill-withdrawal reflex ofrlply- the S-K+channel and a transient K+ channel &). In addition, sia californica. This facilitation is accompanied by a decrease 5-HT increases a voltage-dependent, nifedipine-sensitive in a background current, the S-K+ current (Klein et al., 1982; Ca*+ current. In this article we show that, while the effect on Siegelbaumet al., 1982) as well as a transient K+ current, I,, the S-K+ channel is mediated exclusively by CAMP, the effect (Baxter and Byrne, 1990; Goldsmith and Abrams, 1992; Hoch- on the Ca*+ current can be mimicked by phorbol 12,13-di- ner and Kandel, 1992). The effect on the S-K+ current and on butyrate (PDBu) as well as by intracellular injection of CAMP. I,, can be simulated by increasing the intracellular level of We then use specific blockers of protein kinase C (PKC) and CAMP or of the CAMP-dependent protein kinase A (PKA). In the CAMP-dependent protein kinase A (PKA) to examine the addition to thesetwo K+ currents, Edmondset al. (1987) found roles of PKC and PKA in mediating the effect of 5-HT on the that 5-HT also modulates a slowly inactivating component of nifedipine-sensitive Ca*+ current. the Ca*+ current. Interestingly, modulation of this slowly in- We find that H-7, a kinase inhibitor that appears to inhibit activating current doesnot contribute directly to the facilitation PKC more effectively than PKA in intact Ap/ysia neurons, of transmitter release(see Edmonds et al., 1990). reverses the increase in the Ca*+ current produced by PDBu. Since in addition to increasing intracellular CAMP (Bemier Moreover, H-7 partially blocks the effect of 5-HT on the Ca2+ et al., 1982) 5-HT also causestranslocation of an isoform of current without affecting the decrease in the S-K+ current. protein kinase C (PKC, Braha et al., 1990; Chirardi et al., 1990; A more specific PKC inhibitor (the 19-31 pseudosubstrate Sacktor and Schwartz, 1990), we examined the possiblerole of of PKC) also partially blocks the increase in the Ca*+ current CAMP-dependent phosphorylation via PKA and of Ca*+-phos- produced by 5-HT, suggesting that this increase is mediated pholipiddependent phosphorylation via PKC on the seroto- by PKC. Rp-CAMPS, a specific blocker of PKA, did not block nergic modulation of this Ca*+ current. For comparison,we also the increase in the Ca*+ current produced by 5-HT, sug- examined another representative current, the S-K+ current. We gesting that the effect of 5-HT on this current may be me- find that the S-K+ current is modulated only by the CAMP- diated to only a small extent by PKA. The effect of 5-HT on dependent PKA (for parallel data on I,,, seeHochner and Kan- the S-K+ current and the Ca*+ current can also be separated del, 1992, and Goldsmith and Abrams, 1992). It is not affected on basis of the time course of their appearance. The fact by the activation of PKC, nor is it blocked by H-7. By contrast, that the decrease in the S-K+ current precedes the increase 5-HT modulation of the slowly inactivating Ca*+ current can in Ca*+ current suggests that there may be a temporal dif- be simulated by both CAMP and phorbol esters.However, the ference in the activation of the two kinase systems. modulation of this current by 5-HT is not blocked by inhibiting [Key words: protein kinase C (PKC), protein kinase A (PKA), PKA but is partially blocked by inhibiting PKC. Taken together, Aplysia sensory neurons, phorbol esters, calcium current, the data suggestthat whereas the serotonergic modulation of second messengers, current modulation] the S-K+ current is mediated by PKA, the modulation of the Ca2+current seemsto involve primarily PKC. Materials and Methods Cell preparation. Experimentswere done on primary cell culturespre- Received Sept. 3, 1992; revised Oct. 28, 1992; accepted Nov. 2, 1992. paredby the methoddescribed by Schacherand Proshansky(1983) and This work was supported in part by the Howard Hughes Medical Institute and by a grant to M.K. from the National Institutes of Mental Health (MH45397). Rayport and Schacher (1986). Pleural and abdominal ganglia were dis- We ako thank Bruce Kemp from Melbourne, Australia, for providing the peptide sected from adult Aplysiu (100-l 50 gm) anesthetized with 50-75 ml of inhibitor for PKC, and Binyamin Hochner for his comments on the manuscript. isotonic MgCI,. The ganglia were incubated for 2-2.5 hr at 34°C in 1% Corresnondence should be addressed to Dr. Eric R. Kandel. Center for Neu- protease type IX (Sigma) in L15 (modified for ApIysia; Plow Labora- robiology and Behavior, College of Physicians and Surgeons of Columbia Uni- tories). After rinsing, the ganglia were transferred to a Sylgard dish versity, 722 West 168th Street, New York, NY 10032. containing 1% fetal calf serum in L15, or 50% L15 and 50% filtered Copyright 0 1993 Society for Neuroscience 0270-6474/93/131839-13$05.00/O Aplysia hemolymph. The ganglia were pinned and desheathed, and in- 1840 Braha et al. * PKA and PKC Contribute in 5-HT Modulation of I=. dividual cells were removed with a micropipette. The cells were trans- configuration (Hamill et al., 198 1). The pipette solution contained (in ferred to polylysine-coated dishes containing 50% L15 and 50% he- mM) 450 CsCl, 2 MgCI,, 100 HEPES, 5 Na,ATP, 1 Na,GTP, 10 reduced molymph. In some voltage-clamp experiments, cells were plated on glutathionine, 100 glucose, and 10 EGTA, pH 7.3; extracellular solution uncoated dishes and grown in modified L15 alone. These cultures grew contained 450 TEA-Br (Kodak), 10 KCI, -11 CaCl,, 55 MgCl,, and 10 fewer processes, which was advantageous for voltage-clamp experi- HEPES. DH 7.5. In experiments where the vohaae resnonse to 5-HT ments. was examined, KC1 was used instead of CsCl in thepipette solution and Materials were purchased from Sigma, unless otherwise stated. Se- the extracellular medium had the relative ionic composition of normal rotonin creatinine sulfate (5-HT), was dissolved in distilled water to Aplysiu saline, with all the concentrations reduced by 5%. In both types form a stock solution of 10 mM, and diluted in the bath solution before of experiments, two cells less than 200 PM apart were monitored at the application. It was usually applied to the bath to give a final concen- same time; the pipette recording from one cell contained intracellular tration of 10 PM. The inactive a-isomer and the active &isomer of solution with 500 PM Rp-CAMPS, while the other contained the same phorbol dibutyrate (PDBu; LC Services Corp.) were dissolved in di- solution without Rp-CAMPS. Responses of the two cells to the same methyl sulfoxide (DMSO) to form a stock solution of lo-100 mM and application of 5-HT were compared. kept at -20°C. Before each experiment they were diluted with the re- A synthetic peptide inhibitor of PKC (PKCI), a 13 amino acid (19- cording medium and applied directly to the bath to give the specified 3 1) pseudosubstrate (House and Kemp, 1987), was kindly provided by concentration. The membrane-permeable CAMP analog 8-(4-chloro- Bruce Kemp (Melbourne, Australia). Stock solutions (10 mM) of the phenylthio)-CAMP (CPTcAMP) was dissolved in distilled water at 5 inhibitor in distilled water were aliquoted and kept at -70°C. The mM and applied to the bath to give a final concentration of 100 PM in peptide inhibitor was pressure injected into the sensory cells from mi- conjunction with 100 WM isobutylmethylxanthine (IBMX) in 0.1% cropipettes containing 0.5 M K-acetate with 200-500 PM PKCI and 0.2% DMSO. Another membrane-permeable analog of CAMP, L-benzylthio- fast green. The PKCI was injected into the cell by applying repeated 20 CAMP (BTcAMP), was dissolved in distilled water at 10 mM and applied msec pulses of 10 psi. Injection was monitored by visual detection of to the bath to give final concentration of 100-200 PM. I-(5-Isoquino- fast green in the cell. Experiments were discarded in which the pressure linesulfonyl)-2-methylpiperazine dihydrochloride (H-7) purchased from injection caused a large increase in leakage (monitored by 20 mV hy- Seikagaku America, Inc., was dissolved in distilled water and aliquots perpolarizing pulses). were lyophilized and kept at 4°C. Before each experiment it was dis- In some experiments where a fast exchange of solutions was required solved in the recording medium and applied at final concentrations of we applied the various drugs using a multibarrel microperfusion system 100, 200, or 400 FM. The same aliquots were used for the biochemical similar to the one used in Boll and Lux (1985).
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