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Department of Pharmacology, University of Iowa, College of Medicine, Iowa City, Iowa 52242-1109
Regulators of G protein signaling (RGS) proteins bind to the
subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among G, Gβ
, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein–coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing 1B, 2, and β3 Ca channel subunits and m2 receptors, carbachol (1 µM) inhibited whole-cell currents by 
80% compared with only 55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose–response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being 18 nM in control cells vs. 150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some G subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein–modulated Ca channels.
Key Words: muscarinic receptors • voltage-gated calcium channel • neurosecretion • presynaptic inhibition • regulator of G protein signaling proteins
Abbreviations: CCh, carbachol; EGFP, enhanced green fluorescent protein; GAP, GTPase-accelerating protein; GIRK, G protein–gated inward rectifier K; HEK, human embryonic kidney; RGS, regulator of G protein signaling
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