Abstract

Ca_V2.3 subunits are expressed in neuronal and neuroendocrine cells where they are believed to form native R-type Ca²⁺ channels. Although R-type currents are involved in triggering neurotransmitter and hormone secretion, little is known about their modulation. Previous studies have shown that muscarinic acetylcholine receptors evoke both inhibition and stimulation of Ca_V2.3. Muscarinic inhibition of Ca_V2.3 is mediated by Gβγ subunits, whereas stimulation is mediated by pertussis toxin-insensitive Gα subunits. In the present study, we compared modulation of Ca_V2.3 by the three Gαq/11-coupled muscarinic receptors (M1, M3, and M5). Our data indicate that these receptors trigger comparable stimulation of Ca_V2.3. The signaling pathway that mediates stimulation was meticulously analyzed for M1 receptors. Stimulation is blocked by neutralizing antibodies directed against Gαq/11, coexpression of the regulatory domain of protein kinase Cδ (PKCδ), preactivating PKC with phorbol ester, or pharmacological suppression of PKC with bisindolylmaleimide I. Stimulation of Ca_V2.3 is Ca²⁺-independent and insensitive to 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö 6976), a specific inhibitor of Ca²⁺-dependent PKC isozymes. These results indicate that muscarinic stimulation of Ca_V2.3 involves signaling by Gαq/11, diacylglycerol, and a Ca²⁺-independent PKC. In contrast to stimulation, the magnitude of Ca_V2.3 inhibition depended on receptor subtype, with M3 and M5 receptors producing much larger Ca_V2.3 inhibition than M1 receptors. Interestingly, muscarinic inhibition of Ca_V2.3 was notably enhanced during pharmacological suppression of PKC, suggesting the presence of cross-talk between Gβγ-mediated inhibition and PKC-mediated stimulation of R-type channels similar to that described previously for N-type channels.