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

I-Shan Chen,Kazuharu Furutani,Yoshihisa Kurachi

doi:10.1038/s41598-017-05128-z

Abstract

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.

Highlights

Membrane potential controls physiological events through regulating a variety of protein functions, including modulation of ligand binding and signal transduction in G-protein-coupled receptors (GPCRs)[1,2,3,4,5]
The current decay during hyperpolarization was not observed in the absence of RGS4 (Fig. 1e,f), suggesting that RGS4 is a crucial mediator in the hyperpolarization-induced desensitization of the gated inwardly rectifying K+ (GIRK) channel
Since the intracellular loop 3 of the M2 muscarinic receptor is involved in the voltage-dependence of ACh binding[3], we examine the roles of the intracellular loop 3 in the voltage-dependence of RGS4 modulation of pilocarpine-induced GIRK current

Summary

Introduction

Membrane potential controls physiological events through regulating a variety of protein functions, including modulation of ligand binding and signal transduction in G-protein-coupled receptors (GPCRs)[1,2,3,4,5]. One of the important regions for the voltage-dependent behavior of M2 muscarinic receptor toward its agonist is intracellular loop 3, which is the key motif responsible for G-protein interaction[3, 12, 14, 15]. There is no evidence whether the voltage sensor function of the M2 muscarinic receptor via DRY motif or the intracellular loop 3 can regulate its downstream G-protein signaling regulators. Pilocarpine promotes the RGS4-mediated inhibition of M2 muscarinic receptor-activated G-protein signaling, leading to a smaller G-protein-gated inwardly rectifying K+ (GIRK) current than that of ACh18. The DRY motif and the intracellular loop 3 at the M2 muscarinic receptor are candidates for the structural determinants which contribute to the voltage-dependence of RGS4

Methods

Results

Conclusion