Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes
Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes
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DOI：
10.1093/cvr/cvp039
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发表时间：
2009-04-01
影响因子：
10.8
通讯作者：
Hu, Keli
中科院分区：
文献类型：
--
作者：
Garg, Vivek;Jiao, Jundong;Hu, Keli
心肌中的K-ATP通道是缺血时细胞兴奋性和动作电位的重要调节因子。然而，对这些通道的亚细胞定位及其调控知之甚少。本研究以成年和新生大鼠心肌细胞为研究对象，探讨小凹在心肌细胞K-ATP通道调节中的作用。采用密度梯度离心法、免疫印迹法、免疫共沉淀法和免疫荧光共聚焦显微镜技术，结合全细胞电压钳记录和siRNA基因沉默技术进行亚细胞分离。我们通过细胞分离、超速离心和Western blotting检测到心肌细胞质膜上的K-ATP通道主要分布在小窝蛋白-3富含的微区中。免疫荧光共聚焦显微镜显示K-ATP通道成孔亚基Kir6.2和小窝蛋白-3沿细胞膜广泛共存。心肌细胞的免疫共沉淀显示Kir6.2、腺苷A(1)受体和小窝蛋白-3显著相关。此外，全细胞电压钳研究表明，腺苷A(1)受体介导的K-ATP通道激活可被甲基-β-环糊精或小干扰RNA破坏小窝而基本消除，而吡那地尔诱导的K-ATP通道激活不受影响。这种微域关联对于腺苷受体介导的心肌细胞K-ATP通道的调节是必不可少的。
ATP-sensitive potassium (K-ATP) channels in the heart are critical regulators of cellular excitability and action potentials during ischaemia. However, little is known about subcellular localization of these channels and their regulation. The present study was designed to explore the potential role of caveolae in the regulation of K-ATP channels in cardiac ventricular myocytes.Both adult and neonatal rat cardiomyocytes were used. Subcellular fractionation by density gradient centrifugation, western blotting, co-immunoprecipitation, and immunofluorescence confocal microscopy were employed in combination with whole-cell voltage clamp recordings and siRNA gene silencing. We detected that the majority of K-ATP channels on the plasma membrane of cardiac myocytes were localized in caveolin-3-enriched microdomains by cell fractionation and ultracentrifugation followed by western blotting. Immunofluorescence confocal microscopy revealed extensive colocalization of K-ATP channel pore-forming subunit Kir6.2 and caveolin-3 along the plasma membrane. Co-immunoprecipitation of cardiac myocytes showed significant association of Kir6.2, adenosine A(1) receptors, and caveolin-3. Furthermore, whole-cell voltage clamp studies suggested that adenosine A(1) receptor-mediated activation of K-ATP channels was largely eliminated by disrupting caveolae with methyl-beta-cyclodextrin or by small interfering RNA, whereas pinacidil-induced K-ATP activation was not altered.We demonstrate that K-ATP channels are localized to caveolin-enriched microdomains. This microdomain association is essential for adenosine receptor-mediated regulation of K-ATP channels in cardiac myocytes.