Abstract

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 N-type 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 3′,5′-phosphorothioate, 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.