Mechanism of action of Gq to inhibit Gβγ modulation of Cav2.2 calcium channels:: Probed by the use of receptor-Gα tandems

The stable. interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the alpha(2A) adrenergic receptor (alpha(2A)-R) and pertussis toxin-resistant mutants of different Galpha subunits of heterotrimeric G-proteins. Both alpha(2A)-R-Galpha(o) and alpha(2A)-R-Galpha(i) proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (Ca(v)2.2) similar to the wild-type alpha(2A)-R when expressed in COS-7 cells. The interaction of G(q) with the G(i/o) signaling pathways was studied by expressing either Galpha(q) or a chimeric construct based on Galpha(q) containing the last five amino acids of Galpha(z), which is activated by alpha(2A)-R. It was found that Galpha(qz5) activated by the wild-type alpha(2A)-R inhibited Ca(v)2.2 currents in a voltage-independent fashion. Furthermore, Galpha(qz5) counteracted the voltage-dependent inhibition resulting from alpha(2A)-R-Galpha(o) activation. We subsequently investigated the basis for the behavior of Galpha(qz5). Our evidence suggests that this occurs as a result of a downstream effect of activation of Galpha(qz5) because it was blocked by C-terminal construct of phospholipase Cbeta1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of Galpha(qz5) in alpha(2A)-R-Galpha(o)-transfected cells. Conversely, cells expressing both alpha(2A)-R-Galpha(o) and Galpha(qz5) 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.