Mechanism of Action of Gq to Inhibit Gβγ Modulation of CaV2.2 Calcium Channels: Probed by the Use of Receptor-Gα Tandems

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Abstract
The stable interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the α2A adrenergic receptor (α2A-R) and pertussis toxin-resistant mutants of different Gα subunits of heterotrimeric G-proteins. Both α2A-R-Gαoand α2A-R-Gαi proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (CaV2.2) similar to the wild-type α2A-R when expressed in COS-7 cells. The interaction of Gq with the Gi/o signaling pathways was studied by expressing either Gαq or a chimeric construct based on Gαqcontaining the last five amino acids of Gαz, which is activated by α2A-R. It was found that Gαqz5activated by the wild-type α2A-R inhibited CaV2.2 currents in a voltage-independent fashion. Furthermore, Gαqz5 counteracted the voltage-dependent inhibition resulting from α2A-R-Gαoactivation. We subsequently investigated the basis for the behavior of Gαqz5. Our evidence suggests that this occurs as a result of a downstream effect of activation of Gαqz5 because it was blocked by C-terminal construct of phospholipase Cβ1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of Gαqz5 in α2A-R-Gαo-transfected cells. Conversely, cells expressing both α2A-R-Gαo and Gαqz5 exhibited a partial restoration of voltage-dependent inhibition in the presence of the PKC inhibitor bisindolylmaleimide I (GF 109203X). The potential sites of phosphorylation are discussed.

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