PROTEIN-KINASE-A REGULATES INHIBITION OF N-TYPE AND P Q-TYPE CALCIUM CHANNELS BY ETHANOL IN PC12 CELLS/

Ethanol inhibits L-type Ca++ channels, but little is known about its e ffect on other voltage-gated Ca++ channels. To examine non-L-type chan nels we used nerve growth factor-differentiated PC12 cells treated wit h the L channel blocker nifedipine. Using selective Ca++ channel antag onists, we found that N-type and P/Q-type channels mediate most of the remaining depolarization-evoked Ca++ rise. Ethanol (10-150 mM) inhibi ted depolarization-induced rises in intracellular Ca++ with maximal in hibition of 46% achieved using 50 mM ethanol. Inhibition was time depe ndent, requiring at least 8 min to develop fully. Ethanol did not alte r Ca++ mobilization, sequestration, extrusion or capacitative entry. S p-adenosine cyclic 3',5'-phosphorothioate, a specific activator of pro tein kinase A (PKA), blocked inhibition by ethanol, whereas the protei n kinase C activator phorbol 12-myristate, 13-acetate did not. Okadaic acid, an inhibitor of protein phosphatases type-1 and type-2A, also b locked 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 antagoni zed by activating PKA. The findings suggest the sensitivity of these c hannels to ethanol is regulated by a phosphoprotein that is a substrat e for PKA and protein phosphatase type-2A.