Creatine kinase is physically associated with the cardiac ATP-sensitive k+ channel in vivo
Creatine kinase is physically associated with the cardiac ATP-sensitive k+ channel in vivo
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DOI：
10.1096/fj.01-0446fje
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发表时间：
2001-11-01
期刊：
影响因子：
4.8
通讯作者：
Jovanovic, A
中科院分区：
文献类型：
--
作者：
Crawford, RM;Ranki, HJ;Jovanovic, A
心脏肌层ATP敏感K(+) （K(ATP)）通道由Kir6.2和SUR2A亚基组成，将细胞的代谢状态与膜的兴奋性耦合在一起。基于先前的功能研究，我们假设肌酸激酶（CK）可能是肌上皮K（ATP）通道蛋白复合物的一部分。内外膜片钳和全细胞膜片钳对豚鼠心肌细胞的电生理实验表明，CK底物对K（ATP）通道活性具有调节作用。用抗sur2抗体免疫沉淀豚鼠心膜后，考马斯蓝染色显示，除Kir6.2和SUR2A外，还有一个类似于48 kDa的多肽。Western blotting分析证实了假定的Kir6.2和SUR2A的性质，而基质辅助激光解吸/电离飞行时间质谱分析鉴定了p48 kDa是CK的肌肉形式。此外，在抗sur2a免疫沉淀中发现了CK活性，并且在抗CK抗体和抗sur2a免疫沉淀之间观察到交叉反应性，反之亦然。在重组通道亚基水平上获得的进一步结果表明，CK与SUR2A直接物理关联，而不是Kir6.2亚基。综上所述，这些结果表明CK在体内与SUR2A亚基相关，SUR2A亚基是肌上皮K（ATP）通道蛋白复合物的一个组成部分。
Cardiac sarcolemmal ATP-sensitive K(+) (K(ATP)) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal K(ATP) channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate K(ATP) channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at similar to 48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal K(ATP) channel protein complex.