Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.

Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.