Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.
Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.
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DOI：
10.1074/jbc.m109.093500
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发表时间：
2010-07-23
期刊：
影响因子：
--
通讯作者：
Ma R
中科院分区：
文献类型：
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作者：
Graham S;Ding M;Ding Y;Sours-Brothers S;Luchowski R;Gryczynski Z;Yorio T;Ma H;Ma R
本研究利用TRPC 6异源表达系统研究了H2 O2对TRPC 6通道的影响及其机制。在表达TRPC 6的HEK 293 T细胞中，H2 O2以剂量依赖性方式显著刺激Ca 2+内流。电生理实验表明，H2 O2显著增加TRPC 6通道开放概率和全细胞电流。H2 O2也引起了一个强大的内向电流在A7 r5血管平滑肌细胞，这是几乎取消了敲低TRPC 6使用小干扰RNA。过氧化氢酶显著减弱精氨酸加压素（AVP）诱导的细胞内钙离子内流与TRPC 6和AVP V1受体共转染。N-乙基马来酰亚胺和硫柳汞能够模拟H2 O2响应。二硫苏糖醇或谷胱甘肽减少乙酯显着拮抗的反应。此外，N-乙基马来酰亚胺-和H2 O2-诱导的TRPC 6激活仅在细胞附着的补丁中观察到，但在由内而外的补丁中没有观察到。此外，1-油酰基-2-乙酰基-sn-甘油对TRPC 6的作用在H2 O2存在下显著更大。生物素化试验显示细胞表面TRPC 6响应于H2 O2显著增加。类似地，在用TRPC 6-EGFP转染的细胞中，共聚焦显微镜显示在细胞膜区域和膜附近的荧光强度显著增加。AVP还可增加TRPC 6-EGFP和V1受体共转染细胞表面的荧光强度，这种反应可被过氧化氢酶抑制。这些数据表明，H2 O2通过修饰细胞内蛋白质的巯基来激活TRPC 6通道。这种半胱氨酸氧化依赖性途径不仅本身刺激TRPC 6通道，而且使通道对二酰基甘油敏感，并促进TRPC 6运输到细胞表面。
This study examined the effect of H2O2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H2O2 significantly stimulated Ca2+ entry in a dose-dependent manner. Electrophysiological experiments showed that H2O2 significantly increased TRPC6 channel open probability and whole-cell currents. H2O2 also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca2+ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H2O2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H2O2-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H2O2. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H2O2. Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H2O2 activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.