Cholesterol Inhibits M-type K+ Channels via Protein Kinase C-dependent Phosphorylation in Sympathetic Neurons
Cholesterol Inhibits M-type K+ Channels via Protein Kinase C-dependent Phosphorylation in Sympathetic Neurons
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DOI：
10.1074/jbc.m109.048868
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发表时间：
2010-04-02
影响因子：
4.8
通讯作者：
Cho, Hana
中科院分区：
文献类型：
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作者：
Lee, Seul-Yi;Choi, Hyun-Kyung;Cho, Hana
M型钾通道（KCNQ）在调节神经元动作电位放电中起重要作用。本文应用膜片钳技术研究了胆固醇对颈上神经节（上级）交感神经元M电流的影响。M电流被抑制在一个剂量依赖性的方式与甲基-β-环糊精-胆固醇复合物的胆固醇负载。当通过在移液管溶液中包括空的甲基-β-环糊精来恢复膜胆固醇水平时，这种效应被阻止。用AMP-PNP代替ATP透析细胞可阻止胆固醇对M电流的作用。蛋白激酶C（PKC）抑制剂，calphostin C，废除胆固醇诱导的抑制，而PKC激活剂，PDBu，模仿胆固醇对M电流的抑制。体外激酶试验表明，M通道KCNQ 2亚基可被PKC磷酸化。一个KCNQ 2突变体，是由PKC磷酸化缺陷未能显示电流抑制不仅由PDBu，但也由胆固醇。这些结果表明，胆固醇诱导的M电流的抑制介导的PKC磷酸化。PDBu和胆固醇对M电流的抑制被PIP 2负载完全阻断，表明PIP 2通道相互作用的减少是PKC介导的磷酸化对M通道抑制的基础。我们的结论是胆固醇通过PKC激活特异性调节SCG神经元的M电流。
M-type (KCNQ) potassium channels play an important role in regulating the action potential firing in neurons. Here, we investigated the effect of cholesterol on M current in superior cervical ganglion (SCG) sympathetic neurons, using the patch clamp technique. M current was inhibited in a dose-dependent manner by cholesterol loading with a methyl-beta-cyclodextrin-cholesterol complex. This effect was prevented when membrane cholesterol level was restored by including empty methyl-beta-cyclodextrin in the pipette solution. Dialysis of cells with AMP-PNP instead of ATP prevented cholesterol action on M currents. Protein kinase C (PKC) inhibitor, calphostin C, abolished cholesterol-induced inhibition whereas the PKC activator, PDBu, mimicked the inhibition of M currents by cholesterol. The in vitro kinase assay showed that KCNQ2 subunits of M channel can be phosphorylated by PKC. A KCNQ2 mutant that is defective in phosphorylation by PKC failed to show current inhibition not only by PDBu but also by cholesterol. These results indicate that cholesterol-induced inhibition of M currents is mediated by PKC phosphorylation. The inhibition of M currents by PDBu and cholesterol was completely blocked by PIP2 loading, indicating that the decrease in PIP2-channel interaction underlies M channel inhibition by PKC-mediated phosphorylation. We conclude that cholesterol specifically regulates M currents in SCG neurons via PKC activation.