Differential Modulation of Ca V 2.3 Ca 2ϩ Channels by G␣q/11-Coupled Muscarinic Receptors

Abstract

ABSTRACT Ca V 2.3 subunits are expressed in neuronal and neuroendocrine cells where they are believed to form native R-type Ca 2ϩ 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 V 2.3. Muscarinic inhibition of Ca V 2.3 is mediated by G␤␥ subunits, whereas stimulation is mediated by pertussis toxininsensitive G␣ subunits. In the present study, we compared modulation of Ca V 2.3 by the three G␣q/11-coupled muscarinic receptors (M1, M3, and M5). Our data indicate that these receptors trigger comparable stimulation of Ca V 2.3. The signaling pathway that mediates stimulation was meticulously analyzed for M1 receptors. Stimulation is blocked by neutralizing antibodies directed against G␣q/11, coexpression of the regulatory domain of protein kinase C␦ (PKC␦), preactivating PKC with phorbol ester, or pharmacological suppression of PKC with bisindolylmaleimide I. Stimulation of Ca V 2.3 is Ca 2ϩ -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 Ca 2ϩ -dependent PKC isozymes. These results indicate that muscarinic stimulation of Ca V 2.3 involves signaling by G␣q/11, diacylglycerol, and a Ca 2ϩ -independent PKC. In contrast to stimulation, the magnitude of Ca V 2.3 inhibition depended on receptor subtype, with M3 and M5 receptors producing much larger Ca V 2.3 inhibition than M1 receptors. Interestingly, muscarinic inhibition of Ca V 2.3 was notably enhanced during pharmacological suppression of PKC, suggesting the presence of cross-talk between G␤␥-mediated inhibition and PKC-mediated stimulation of R-type channels similar to that described previously for N-type channels