Li Ka Shing Institute of Health Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China.
J Biol Chem. 2011 Jun 3;286(22):19439-45. doi: 10.1074/jbc.M110.210294. Epub 2011 Apr 12.
cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 μm 8-Br-cAMP, a cell-permeable analog of cAMP, elicited Ca(2+) increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced Ca(2+) increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.
cAMP 是一种重要的第二信使，在活细胞中执行多种生理功能。在这项研究中，我们研究了 cAMP 对表达 TRPC6 的 HEK293 细胞和肾小球系膜细胞中典型的 TRPC6 通道的影响。结果表明，细胞可通透的 cAMP 类似物 500μm 8-Br-cAMP 在表达 TRPC6 的 HEK293 细胞中引起 Ca(2+)增加，并在全细胞水平上刺激阳离子电流。在存在 PI3K 抑制剂wortmannin 和 LY294002 或 MEK 抑制剂 PD98059、U0126 和 MEK 抑制剂 I 的情况下，cAMP 的作用减弱。8-Br-cAMP 还诱导 MEK 和 ERK1/2 的磷酸化。在 ERK1/2 磷酸化位点（S281G）将丝氨酸转化为甘氨酸可消除 cAMP 对 TRPC6 的激活，这通过全细胞和细胞贴附式单通道膜片钳记录来确定。基于一组药理学抑制剂或激活剂的实验表明，cAMP 对 TRPC6 的作用不是由 PKA、PKG 或 EPAC（cAMP 激活的交换蛋白）介导的。全内反射荧光反射显微镜显示，8-Br-cAMP 不会改变 TRPC6 向质膜的转运。我们还发现，在肾小球系膜细胞中，胰高血糖素诱导的 Ca(2+)增加是通过 cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 信号通路介导的。总之，这项研究揭示了一种新的 TRPC6 激活机制，即 cAMP 通过 PI3K-PKB-MEK-ERK1/2 信号通路激活 TRPC6。
