In the present study, we investigated the role of endogenous regulator of G protein signaling(RGS) proteins in G protein-mediated ion channel modulation. To this end, GαoA was rendered PTX-insensitive(C351G; PTX-i), and both PTX-and RGS- insensitive(G184S:C351G; PTX/RGS-i)1,2 by site-directed mutagenesis. The mutants were coexpressed with Gβ1γ2 subunits by intranuclear injection of CMV-driven plasmids into rat sympathetic neurons. Voltage-gated Ca2+ and GIRK(1 and 4) currents were measured using the whole-cell variant of the patch clamp technique. After overnight treatment with PTX to suppress natively expressed Gα subunits, voltage-dependent Ca2+ current inhibition by NE was reconstituted in neurons expressing either PTX-i or PTX/RGS-i GαoA. When compared with neurons expressing PTX-i GαoA, the steady-state concentration-response relationships for NE-induced Ca2+ current inhibition were shifted to lower concentrations in neurons expressing PTX/RGS-i GαoA. In addition to an increase in agonist potency, the on/off kinetics of the Ca2+ current inhibition were significantly retarded. When compared with PTX-i GαoA expressing neurons, expression of the PTX/RGS-i GαoA resulted in: i) a prolonged lag phase preceding GIRK activation, ii) retarded activation/deactivation kinetics, and iii) ablation of the acute phase of GIRK current desensitization. Surprisingly, coexpression of RGS8 with PTX/RGS-i GαoA significantly shortened the prolonged lag phase without accelerating GIRK current deactivation. This phenomenon was found to arise from the N-terminus, but not the core domain of RGS8. Taken together, these findings suggest that endogenous RGS proteins i) play essential roles in regulating the strength and life time of G protein signaling, and ii) may influence receptor/effector coupling independently of their ability to augment Gα GTPase activity.
Source: Korean Journal of Physiology & Pharmacology.2000 Oct;4(Suppl):S7-S8
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