Activation of M1 muscarinic acetylcholine receptors stimulates the formation of a multiprotein complex centered on TRPC6 channels
Activation of M1 muscarinic acetylcholine receptors stimulates the formation of a multiprotein complex centered on TRPC6 channels
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DOI：
10.1074/jbc.m500429200
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发表时间：
2005-09-09
影响因子：
4.8
通讯作者：
Saffen, D
中科院分区：
文献类型：
--
作者：
Kim, JY;Saffen, D
在这项研究中，我们表明，刺激M1毒蕈碱乙酰胆碱受体（mAChRs）激活内源性瞬时受体电位典型，亚型6（TRPC 6），通道在神经元PC 12 D细胞。TRPC 6通道的激活与含有M1 mAChR、TRPC 6通道和蛋白激酶C（PKC）的多蛋白复合物的形成相关。M1 mAChR-TRPC 6-PKC复合物的形成是短暂的，在刺激M1 mAChR后2分钟达到最高水平。复合物中的PKC磷酸化羧基末端结构域中保守丝氨酸残基上的TRPC 6（TRPC 6A同种型中的Ser（768）和TRPC 6 B同种型中的Ser（714））。亲免素FKBP 12、磷酸酶钙调磷酸酶和钙调蛋白也在M1 mAChR活化后被募集到M1 mAChR-TRPC 6-PKC复合物中，并在M1 mAChR和PKC解离后与TRPC 6通道保持稳定缔合。FKBP 12、钙调磷酸酶和钙调蛋白与TRPC 6通道的结合通过以下方式阻断：1）抑制PKC; 2）通道中PKC磷酸化位点（Ser（7168/714））突变;或3）用FK 506或雷帕霉素（直接结合FKBP 12的免疫抑制剂）预处理。FKBP 12结合的抑制阻断TRPC 6通道的去磷酸化和M1 mAChR的解离，而不影响PKC的解离。钙调磷酸酶抑制剂环孢菌素A也阻断TRPC 6的去磷酸化，并防止M1 mAChR的解离。总之，这些结果表明，激活的TRPC 6通道形成了一个动态的多蛋白复合物，包括PKC和钙调神经磷酸酶，分别磷酸化和去磷酸化的通道的中心。PKC对TRPC 6通道的磷酸化是FKBP 12结合所必需的，而FKBP 12又是钙调磷酸酶和钙调蛋白结合所必需的。随后通过钙调神经磷酸酶的通道的去磷酸化是M1 mAChRs解离所必需的。
In this study we showed that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) activates endogenous transient receptor potential-canonical, subtype 6 (TRPC6), channels in neuronal PC12D cells. Activation of TRPC6 channels is correlated with the formation of a multiprotein complex containing M1 mAChRs, TRPC6 channels, and protein kinase C (PKC). Formation of the M1 mAChR-TRPC6-PKC complex is transient, with highest levels reached similar to 2 min after stimulation of M1 mAChRs. PKC in the complex phosphorylates TRPC6 on a conserved serine residue in the carboxyl-terminal domain (Ser(768) in the TRPC6A isoform and Ser(714) in the TRPC6B isoform). The immunophilin FKBP12, the phosphatase calcineurin, and Ca2+-binding protein calmodulin are also recruited to the M1 mAChR-TRPC6-PKC complex following activation of M1 mAChRs and remain stably associated with the TRPC6 channels after M1 mAChRs and PKC have disassociated. Binding of FKBP12, calcineurin, and calmodulin to TRPC6 channels is blocked by the following: 1) inhibition of PKC; 2) mutation of the PKC phosphorylation site (Ser(7168/714)) in the channels; or 3) pretreatment with FK506 or rapamycin, immunosuppressants that directly bind FKBP12. Inhibition of FKBP12 binding blocks the dephosphorylation of TRPC6 channels and the disassociation of M1 mAChRs, without affecting disassociation of PKC. The calcineurin inhibitor cyclosporin A also blocks the dephosphorylation of TRPC6 and prevents the disassociation of M1 mAChRs. Together, these results show that activated TRPC6 channels form the center of a dynamic multiprotein complex that includes PKC and calcineurin, which respectively phosphorylate and dephosphorylate the channels. Phosphorylation of the TRPC6 channels by PKC is required for the binding of FKBP12, which in turn is required for the binding of calcineurin and calmodulin. Subsequent dephosphorylation of the channels by calcineurin is required for the disassociation of M1 mAChRs.