Department of Cell and Systems Physiology, University of Occupational and Environmental Health School of Medicine, Kitakyushu, 807-8555, Japan.
J Physiol. 2017 Nov 15;595(22):6851-6867. doi: 10.1113/JP275039. Epub 2017 Oct 19.
The muscarinic acetylcholine receptor (mAChR)-mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K channel)-related acid-sensitive K (TASK)1 channels. In this study we focused on the molecular mechanism of mAChR-mediated inhibition of TASK1 channels. Exposure to muscarine resulted in a clathrin-dependent endocytosis of TASK1 channels following activation of the muscarinic M receptor (M R). This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non-receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1. The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin-dependent manner in response to M R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.
Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK-related acid-sensitive K (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR-mediated inhibition. TASK1-like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine-induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M antagonist, and the dose-response curves for muscarinic agonist-induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine-induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1-like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M R stimulation results in internalization of TASK1 channels through the PLC-PKC-Src pathway with the consequent phosphorylation of tyrosine and that this M R-mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.
毒蕈碱型乙酰胆碱受体（mAChR）介导的大鼠肾上腺髓质细胞兴奋性增加至少部分是由于 TWIK（弱内向整流钾通道中的串联 P 结构域）相关酸敏感钾（TASK）1 通道的抑制。在这项研究中，我们专注于 mAChR 介导的 TASK1 通道抑制的分子机制。毒蕈碱的作用导致 TASK1 通道的网格蛋白依赖性内吞作用，紧随其后的是毒蕈碱 M 受体（M R）的激活。这种 mAChR 信号的内吞作用依次由磷脂酶 C（PLC）、蛋白激酶 C（PKC）介导，然后是非受体酪氨酸激酶Src，导致 TASK1 的酪氨酸磷酸化。目前的结果表明，TASK1 通道在大鼠 AM 细胞中响应 M R 刺激以网格蛋白依赖性方式发生酪氨酸磷酸化和内吞作用，这种易位至少部分负责 mAChR 对 TASK1 通道的抑制。
在大鼠肾上腺髓质（AM）细胞中，毒蕈碱受体（mAChR）的激活通过抑制 TWIK 相关酸敏感钾（TASK）1 通道诱导去极化。在这里，使用药理学和免疫学方法来阐明这种 mAChR 介导的抑制的分子机制。在分离的大鼠 AM 细胞中，TASK1 样免疫反应（IR）物质主要位于细胞外周，并且其大部分在响应毒蕈碱时被内化。Dynasore，一种动力蛋白抑制剂，抑制毒蕈碱诱导的内向电流和 TASK1 的易位。MT7，一种特异性 M 拮抗剂，抑制毒蕈碱易位，并且毒蕈碱激动剂诱导的易位的剂量反应曲线与 TASK1 电流的毒蕈碱抑制相似。TASK1 通道在 AM 细胞和 PC12 细胞中瞬时与Src 相关，并在响应毒蕈碱刺激时发生酪氨酸磷酸化。内化后，即使 TASK1 通道仍在细胞质中，TASK1 通道也很快去磷酸化。毒蕈碱刺激 0.5 分钟后，细胞质中的 TASK1 样 IR 物质迅速回收至细胞外周，但刺激 10 分钟后则不会。我们得出结论，M R 刺激通过 PLC-PKC-Src 途径导致 TASK1 通道内化，随后发生酪氨酸磷酸化，这种 M R 介导的内化至少部分负责大鼠 AM 细胞中 TASK1 通道的毒蕈碱抑制。
