Shimizu Shunichi, Yoshida Takashi, Wakamori Minoru, Ishii Masakazu, Okada Takaharu, Takahashi Masami, Seto Minoru, Sakurada Katsuhiko, Kiuchi Yuji, Mori Yasuo
Department of Pathophysiology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan.
J Physiol. 2006 Jan 15;570(Pt 2):219-35. doi: 10.1113/jphysiol.2005.097998. Epub 2005 Nov 10.
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) 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 Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) 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 Ca(2+)-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 Ca(2+)-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.
果蝇瞬时受体电位（TRP）蛋白的哺乳动物同源物负责脊椎动物细胞中受体激活的Ca(2+)内流。我们之前报道了细胞内Ca(2+)参与哺乳动物典型瞬时受体电位5（TRPC5）通道的受体介导激活。在此，我们研究了钙调蛋白（细胞内Ca(2+)变化的重要传感器）及其下游级联反应在人胚肾（HEK）293细胞中重组TRPC5通道激活中的作用。用钙调蛋白抑制剂W-13处理后，G蛋白偶联ATP受体刺激诱导的通过TRPC5通道的Ca(2+)内流被消除。Ca(2+)-钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）的抑制剂ML-9和渥曼青霉素，以及MLCK显性负性突变体的表达抑制了TRPC5通道活性，揭示了MLCK在维持TRPC5通道活性中的重要作用。需要注意的是，ML-9损害了TRPC5通道的质膜定位。此外，使用全细胞膜片钳技术测量的TRPC5通道活性受到ML-9的抑制，而在细胞切除的内向外膜片中观察到的TRPC5通道活性不受ML-9影响。一种识别磷酸化肌球蛋白轻链（MLC）的抗体显示，在未刺激条件下，HEK293细胞中磷酸化MLC的基础水平因ML-9而降低。这些发现强烈表明，细胞内Ca(2+)-钙调蛋白组成性激活MLCK，从而通过在MLC磷酸化/去磷酸化平衡控制下促进质膜TRPC5通道分布来维持TRPC5通道活性。
