The M2 muscarinic G-protein-coupled receptor is voltage-sensitive
The M2 muscarinic G-protein-coupled receptor is voltage-sensitive
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DOI：
10.1074/jbc.m301146200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Parnas, H
中科院分区：
文献类型：
--
作者：
Ben-Chaim, Y;Tour, O;Parnas, H
G蛋白偶联受体不被认为表现出电压敏感性。在这里，使用非洲爪蟾卵母细胞，我们表明，M-2毒蕈碱受体（M2 R）是电压敏感的。用m2 R介导的钾通道（GIRK）电流检测m2 R的活性。我们发现，表观亲和力的m2 R对乙酰胆碱（ACh）的去极化后减少。结合实验的[ H-3]乙酰胆碱个别卵母细胞表达m2 R证实了电生理的结果。当GIRK通道通过过表达G β γ亚基或通过注射GTP γ S激活时，在-60 mV和+40 mV下测量的电流之间的比率与GIRK通道的基础活性相同。因此，m2 R的激动剂活化下游的步骤不是电压敏感的。我们进一步发现，与m2 R相比，m1 R的表观亲和力在去极化后增加。我们还发现，[H-3]乙酰胆碱结合到表达m2 R的卵母细胞的电压敏感性大大降低后，百日咳毒素预处理。累积结果表明，m2 R本身是电压敏感的。此外，电压敏感性并不存在于ACh结合位点，相反，它最有可能存在于与G蛋白偶联的受体区域。
G- protein coupled receptors are not considered to exhibit voltage sensitivity. Here, using Xenopus oocytes, we show that the M-2 muscarinic receptor ( m2R) is voltage-sensitive. The m2R- mediated potassium channel ( GIRK) currents were used to assay the activity of m2R. We found that the apparent affinity of m2R toward acetylcholine ( ACh) was reduced upon depolarization. Binding experiments of [ H-3] ACh to individual oocytes expressing m2R confirmed the electrophysiological findings. When the GIRK channels were activated either by overexpression of Gbetagamma subunits or by injection of GTPgammaS, the ratio between the currents measured at -60 mV and + 40 mV was the same as for the basal activity of the GIRK channel. Thus, the steps downstream to agonist activation of m2R are not voltage- sensitive. We further found that, in contrast to m2R, the apparent affinity of m1R was increased upon depolarization. We also found that the voltage sensitivity of binding of [H-3] ACh to oocytes expressing m2R was greatly diminished following pretreatment with pertussis toxin. The cumulative results suggest that m2R is, by itself, voltage- sensitive. Furthermore, the voltage sensitivity does not reside in the ACh binding site, rather, it most likely resides in the receptor region that couples to the G- protein.