Endogenous regulator of G-protein signaling proteins modify N-type calciumchannel modulation in rat sympathetic neurons

Experiments using heterologous overexpression indicate that regulator of G- protein signaling (RGS) proteins play important roles in G beta gamma-media ted ion channel modulation. However, the roles subserved by endogenous RGS proteins have not been extensively examined because tools for functionally inhibiting natively expressed RGS proteins are lacking. To address this voi d, we used a strategy in which G alpha(oA) was rendered insensitive to pert ussis toxin (PTX) and RGS proteins by site-directed mutagenesis. Either PTX -insensitive (PTX-i) or both PTX- and RGS-insensitive (PTX/RGS-i) mutants o f G alpha(oA) were expressed along with G beta(1) and G gamma(2) subunits i n rat sympathetic neurons. After overnight treatment with PTX to suppress n atively expressed G alpha subunits, voltage-dependent Ca2+ current inhibiti on by norepinephrine (NE) (10 mu M) was reconstituted in neurons expressing either PTX-i or PTX/RGS-i G alpha(oA). When compared with neurons expressi ng PTX-i G alpha(oA), the steady-state concentration-response relationships for NE-induced Ca2+ current inhibition were shifted to lower concentration s in neurons expressing PTX/RGS-i G alpha(oA). In addition to an increase i n agonist potency, the expression of PTX/RGS-i G alpha(oA) dramatically ret arded the current recovery after agonist removal. Interestingly, the altera tion in current recovery was accompanied by a slowing in the onset of curre nt inhibition. Together, our data suggest that endogenous RGS proteins cont ribute to membrane-delimited Ca2+ channel modulation by regulating agonist potency and kinetics of G-protein-mediated signaling in neuronal cells.