Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes ENRICHMENT AT THE INTERCALATED DISK
Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes ENRICHMENT AT THE INTERCALATED DISK
复制标题
DOI：
10.1074/jbc.m112.412122
复制
发表时间：
2012-11-30
影响因子：
4.8
通讯作者：
Coetzee, William A.
中科院分区：
文献类型：
--
作者：
Hong, Miyoun;Bao, Li;Coetzee, William A.
心室ATP敏感性钾（K-ATP）通道将细胞内能量代谢与膜的兴奋性和收缩性联系起来。我们最近的蛋白质组学实验确定斑珠蛋白和斑嗜蛋白-2（PKP 2）为推定的K-ATP通道相关蛋白。我们研究了K-ATP通道亚基与连接蛋白的结合是否转化为心肌细胞内的异质亚细胞分布。免疫共沉淀实验证实了大鼠心脏中K-ATP通道与PKP 2和斑珠蛋白之间的物理相互作用。免疫定位实验表明，K-ATP通道亚基（Kir6.2和SUR 2A）在小鼠和大鼠心脏的闰盘上以更高的密度表达，在那里它们与PKP 2和斑珠蛋白共定位。超分辨率显微镜显示，K-ATP通道聚集在纳米距离的连接蛋白。局部K-ATP通道密度，记录在切除的内面向外补丁，是较大的细胞端相比，从细胞中心记录的局部电流。K-ATP通道单一电导，阻断MgATP和激活MgADP，这两个位置之间没有差异。全细胞K-ATP通道电流密度（通过代谢抑制激活）与来自PKP 2单倍不足小鼠的肌细胞中的小40%相似。与切除的补丁的实验表明，K-ATP通道的区域异质性是不存在的PKP 2缺陷小鼠，但K-ATP通道的单位电导和核苷酸的敏感性保持不变。我们的数据表明心肌细胞内K-ATP通道分布的异质性。闰盘上较高的K-ATP通道密度意味着心肌缺血时细胞间连接可能起作用。
Ventricular ATP-sensitive potassium (K-ATP) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K-ATP channel-associated proteins. We investigated whether the association of K-ATP channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K-ATP channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K-ATP channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K-ATP channels are clustered within nanometer distances from junctional proteins. The local K-ATP channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K-ATP channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K-ATP channel current density (activated by metabolic inhibition) was similar to 40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K-ATP channels was absent in the PKP2 deficient mice, but the K-ATP channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K-ATP channel distribution within a cardiac myocyte. The higher K-ATP channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.