Protein Kinase A Regulates Inhibition of N- and P/Q-type Calcium Channels by Ethanol in PC12 Cells

Ethanol inhibits L-type Ca channels, but little is known about its effect on other voltage-gated Ca channels. To examine non-L-type channels we used nerve growth factor-differentiated PC12 cells treated with the L channel blocker nifedipine. Using selective Ca channel antagonists, we found that Ntype and P/Q -type channels mediate most of the remaining depolarization-evoked Ca rise. Ethanol (10–150 mM) inhibited depolarization-induced rises in intracellular Ca with maximal inhibition of 46% achieved using 50 mM ethanol. Inhibition was time dependent, requiring at least 8 min to develop fully. Ethanol did not alter Ca mobilization, sequestration, extrusion or capacitative entry. Sp-adenosine cyclic 39,59phosphorothioate, a specific activator of protein kinase A (PKA), blocked inhibition by ethanol, whereas the protein kinase C activator phorbol 12-myristate, 13-acetate did not. Okadaic acid, an inhibitor of protein phosphatases type-1 and type-2A, also blocked inhibition by ethanol with an IC50 of 3 nM. This was prevented by inhibiting PKA, indicating that the action of okadaic acid was due to increased PKA-mediated phosphorylation. These results indicate that ethanol can inhibit N-type and P/Q-type channels and this is antagonized by activating PKA. The findings suggest the sensitivity of these channels to ethanol is regulated by a phosphoprotein that is a substrate for PKA and protein phosphatase type-2A. Voltage-gated Ca channels mediate Ca entry into neurons and regulate neurotransmitter release, firing patterns, gene expression and differentiation (McClesky, 1994; Olivera et al., 1994; Ghosh and Greenberg, 1995). Several types of Ca channels have been identified with distinct electrophysiological and pharmacological properties (Zhang et al., 1993; Randall and Tsien, 1995). T channels activate at low voltage, inactivate quickly, and are blocked by low concentrations of Ni. L channels are activated by high voltage, inactivate slowly and are blocked by dihydropyridines. N, P and Q channels are activated by high voltage and blocked by the peptide neurotoxins v-conotoxin GVIA (N), v-agatoxin IVA (P and Q) and v-conotoxin MVIIC (N, P and Q). Other channels have also been described that are resistant to organic Ca channel blockers (G1-G3 and R-type channels) (Forti et al., 1994; Randall and Tsien, 1995). Several manifestations of ethanol intoxication and dependence appear due to changes in Ca channel function (Messing and Diamond, 1997). In nerve terminals from rat neurohypophysis and in NGF-differentiated PC12 cells, brief exposure to intoxicating concentrations (10–50 mM) of ethanol inhibits L-type channels by decreasing open channel probability (Wang et al., 1994) and promoting channel inactivation (Mullikin-Kilpatrick and Treistman, 1995). In N1E115 neuroblastoma and NG108–15 neuroblastoma-glioma cells, high concentrations of ethanol (100–300 mM) reduce T-type currents by 15 to 20% (Twombly et al., 1990), and in rat neurohypophysis, 50 to 100 mM ethanol reduces N-type current by 30 to 40% (Wang et al., 1991). In rat Purkinje neurons P-type currents appear insensitive to ethanol (Hall et al., 1994). The effect of ethanol on other types of Ca channels is not known. Protein phosphorylation regulates ion channels (Nestler and Greengard, 1984) and ethanol can alter phosphorylation through actions on signal transduction pathways that regulate protein kinases, particularly cAMP-dependent PKA and PKC (Messing and Diamond, 1997). In some cells (Nagy et al., 1989; Rabin et al., 1993) ethanol stimulates cAMP formation and activates PKA. In rodent hepatocytes (Hoek et al., 1987) and human platelets (Rubin et al., 1988), ethanol Received for publication March 4, 1997. 1 This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism and the Alcoholic Beverage Medical Research Foundation (R.O.M.). ABBREVIATIONS: PKA, protein kinase A; PKC, protein kinase C; GABA, g-aminobutyric acid; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; Sp-cAMPS, Sp-adenosine cyclic 39, 59-phosphorothioate; Rp-cAMPS, Rp-adenosine cyclic 39, 59-phosphorothioate; DMEM, Dulbecco’s modified Eagle’s medium; cAMP, cyclic adenosine monophosphate; EGTA, ethylene glycol bis(b-aminoethyl ester)-N, N9-tetraacetic acid; HEPES, N-[2-hydroxyethyl]piperazine-N9-[2-ethanesulfonic acid]; [Ca]i, intracellular calcium concentration; PP-1 protein phosphatase type-1; PP-2A, protein phosphatase type-2A; PP-2B, protein phosphatase type-2B. 0022-3565/97/2823-1487$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:1487–1495, 1997 1487 at A PE T Jornals on M ay 2, 2017 jpet.asjournals.org D ow nladed from activates phospholipase C to generate the PKC activator diacylglycerol (Nishizuka, 1992). PKA activation appears to be required for inhibition of adenosine transporters by ethanol (Coe et al., 1996), whereas PKC activation is important for inhibition of AMPA/kainate receptor currents (DildyMayfield and Harris, 1995) and enhancement of mouse and bovine GABAA receptor function (Wafford and Whiting, 1992) by ethanol. No studies have identified a role for phosphorylation in the regulation of voltage-gated Ca channels by ethanol. In this study, we examined the effect of ethanol on non-Ltype Ca channels, by measuring depolarization-induced rises in [Ca]i in NGF-differentiated PC12 cells treated with the L channel blocker nifedipine. Our findings indicate that ethanol can inhibit N-type and P/Q-type channels by a mechanism that is antagonized by PKA.