Vol. 63, Issue 4, 832-843, April 2003

Mechanism of Action of G_q to Inhibit G Modulation of Ca_V2.2 Calcium Channels: Probed by the Use of Receptor-G Tandems

Federica Bertaso, Richard J. Ward, Patricia Viard, Graeme Milligan, and Annette C. Dolphin

Department of Pharmacology, University College London, London, United Kingdom (F.B., P.V., A.C.D.); and Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom (R.J.W., G.M.)

The stable interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the _2A adrenergic receptor (_2A-R) and pertussis toxin-resistant mutants of different G subunits of heterotrimeric G-proteins. Both _2A-R-G_o and _2A-R-G_i proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (Ca_V2.2) similar to the wild-type _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 G_q or a chimeric construct based on G_q containing the last five amino acids of G_z, which is activated by _2A-R. It was found that G_qz5 activated by the wild-type _2A-R inhibited Ca_V2.2 currents in a voltage-independent fashion. Furthermore, G_qz5 counteracted the voltage-dependent inhibition resulting from _2A-R-G_o activation. We subsequently investigated the basis for the behavior of G_qz5. Our evidence suggests that this occurs as a result of a downstream effect of activation of G_qz5 because it was blocked by C-terminal construct of phospholipase C1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of G_qz5 in _2A-R-G_o-transfected cells. Conversely, cells expressing both _2A-R-G_o and G_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.