Ca2+–calmodulin‐dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells
Ca2+–calmodulin‐dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells
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DOI：
10.1113/jphysiol.2005.097998
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发表时间：
2006-01
期刊：
影响因子：
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通讯作者：
S. Shimizu;Takashi Yoshida;M. Wakamori;M. Ishii;T. Okada;Masami Takahashi;M. Seto;K. Sakurada;Y. Kiuchi;Y. Mori
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文献类型：
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作者：
S. Shimizu;Takashi Yoshida;M. Wakamori;M. Ishii;T. Okada;Masami Takahashi;M. Seto;K. Sakurada;Y. Kiuchi;Y. Mori
果蝇瞬时受体电位（TRP）蛋白的哺乳动物同源物是脊椎动物细胞中受体激活的Ca2+内流的原因。我们之前报道了细胞内Ca2+参与受体介导的哺乳动物典型瞬时受体电位5 （TRPC5）通道的激活。在这里，我们研究了钙调素（一种重要的细胞内Ca2+变化传感器）及其下游级联反应在人胚胎肾（HEK） 293细胞重组TRPC5通道激活中的作用。Ca2+通过TRPC5通道进入，由G蛋白偶联ATP受体刺激诱导，通过钙调素抑制剂W - 13处理被消除。ML - 9和wortmannin， Ca2+钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）的抑制剂，以及MLCK显性阴性突变体的表达抑制了TRPC5通道活性，揭示了MLCK在维持TRPC5通道活性方面的重要作用。值得注意的是，ML‐9损害了TRPC5通道的质膜定位。此外，使用全细胞贴片钳技术测量的TRPC5通道活性被ML‐9抑制，而在切除的细胞中观察到的TRPC5通道活性，内外贴片不受ML‐9的影响。一种识别磷酸化肌球蛋白轻链（MLC）的抗体显示，在非刺激条件下，HEK293细胞中磷酸化MLC的基础水平被ML‐9降低。这些发现强烈提示细胞内Ca2+ -钙调蛋白在MLC磷酸化/去磷酸化平衡的控制下，通过促进质膜TRPC5通道分布，组成性地激活MLCK，从而维持TRPC5通道活性。
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor‐activated Ca2+ influx in vertebrate cells. We previously reported the involvement of intracellular Ca2+ in the receptor‐mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca2+, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca2+ entry through TRPC5 channels, induced upon stimulation of the G‐protein‐coupled ATP receptor, was abolished by treatment with W‐13, an inhibitor of calmodulin. ML‐9 and wortmannin, inhibitors of Ca2+–calmodulin‐dependent myosin light chain kinase (MLCK), and the expression of a dominant‐negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML‐9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole‐cell patch‐clamp technique was inhibited by ML‐9, whereas TRPC5 channel activity observed in the cell‐excised, inside‐out patch was unaffected by ML‐9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML‐9 in HEK293 cells. These findings strongly suggest that intracellular Ca2+–calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.