Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.
Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.
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DOI：
10.1038/s41598-017-05128-z
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发表时间：
2017-07-21
影响因子：
4.6
通讯作者：
Kurachi Y
中科院分区：
文献类型：
--
作者：
Chen IS;Furutani K;Kurachi Y
膜电位控制M2受体对其配体的反应。膜超极化增加了对完全激动剂乙酰胆碱(ACh)的反应，而降低了对部分激动剂匹罗卡品的反应。我们以前已经证明，G蛋白信号转导(RGS)4蛋白的调节器区分ACh和匹罗卡品的电压依赖性反应；然而，其潜在的机制尚不清楚。在这里，我们发现RGS4参与M2受体介导的信号对匹罗卡品反应的电压依赖行为。此外，我们还揭示了电压依赖性反应背后的M2 M受体的结构决定因素。通过电生理记录表达M_2受体和G蛋白门控内向整流钾通道的非洲爪哇卵母细胞，我们定量了有或没有RGS4对匹罗卡品诱发电流的电压依赖性脱敏作用。超极化导致RGS4所需电流的脱敏，也依赖于匹罗卡品的浓度。M2受体天冬氨酸-精氨酸-酪氨酸基序中带电残基的突变，而不是细胞内环3的突变，显著削弱了RGS4功能的电压依赖性。因此，我们的结果表明，RGS4调制的电压依赖性来源于M2受体。这些结果为膜电位如何通过调节GPCR与下游效应器的通讯而影响G蛋白信号提供了新的见解。
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.
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