A novel GTP‐dependent mechanism of ileal muscarinic metabotropic channel desensitization
A novel GTP‐dependent mechanism of ileal muscarinic metabotropic channel desensitization
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回肠毒蕈碱代谢通道脱敏的新型 GTP 依赖性机制
DOI：
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发表时间：
1996
影响因子：
7.3
通讯作者：
T. Bolton
中科院分区：
文献类型：
--
作者：
A. Zholos;T. Bolton
1通过使用稳定的毒蕈碱激动剂卡巴胆碱（CCh）激活毒蕈碱受体或使用GTP-γS透析细胞，在单个分离的平滑肌细胞中诱发阳离子电流（Icat）。使用膜片钳记录技术在通过酶消化从豚鼠小肠纵肌层获得的细胞中进行了研究。2 Icat仅在毒蕈碱受体或G蛋白被激活时出现，但它具有强烈的电压依赖性。它的激活可以用玻尔兹曼方程来描述。在50 μ mCCh诱发的Icat脱敏过程中，斜率因子k保持不变，而最大电导Gmax缓慢下降，半最大激活电位V1/2在4 min内正移32 mV。（不含GTP或含1 mM GTP的移液器溶液）或细胞内应用GTP-γS（无CCh）时，Icat的大小和电压依赖性相似。然而，与不含GTP的移液器溶液（应用CCh）相比，移液器溶液中存在GTP（应用CCh）时的Icat脱敏较慢，移液器中存在GTP-γS（无CCh）时的Icat脱敏慢得多; Gmax随时间的降低延迟得多，激活曲线的正移受到抑制。向移液器溶液中加入2 mM的GDP-βS，可消除施加的CCh引起的Icat; 50 μm不能阻止Icat的产生，但可显著加速脱敏。4得出结论，卡巴胆碱诱发的阳离子电流的脱敏率是由于细胞中活化的G蛋白浓度下降，这减少了可以打开的通道的最大数量，并将其激活范围转移到更小的负电位。
1 Cationic current (Icat) was evoked in single isolated smooth muscle cells either by activating muscarinic receptors with the stable muscarinic agonist, carbachol (CCh), or by dialysing cells with GTP‐γS. It was studied using patch‐clamp recording techniques in cells obtained by enzymatic digestion from the longitudinal muscle layer of the guinea‐pig small intestine. 2 Icat appears only when muscarinic receptors or G‐proteins are activated, but it is strongly voltage‐dependent. Its activation could be described by the Boltzmann equation. During desensitization of Icat evoked by 50 μm CCh, the slope factor, k, remained constant whereas the maximal conductance, Gmax, slowly decreased and the potential of half‐maximal activation, V1/2, shifted positively by 32 mV during 4 min. 3 At peak response either to extracellular application of CCh (GTP‐free, or 1 mM GTP‐containing, pipette solution) or to intracellular application of GTP‐γS (no CCh), the size and voltage‐dependent properties of Icat were similar. However, Icat desensitization was slower in the presence of GTP (CCh applied) in the pipette solution and much slower with GTP‐γS in the pipette (no CCh) compared to GTP‐free pipette solution (CCh applied); the decrease in Gmax with time was much delayed and the positive shift of the activation curve was inhibited. GDP‐βS added to the pipette solution at 2 mM abolished Icat in response to applied CCh; 50 μm did not prevent Icat generation but significantly accelerated desensitization. 4 It was concluded that the rate of desensitization of the carbachol‐evoked cationic current was due to a decline in the concentration of activated G‐protein in the cell, which reduced the maximum number of channels which could be opened and shifted their activation range to less negative potentials.
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