Lalli M J, Johns D C, Janecki M, Liu Y, O'Rourke B, Marban E
Section of Molecular and Cellular Cardiology, The Johns Hopkins University, School of Medicine, 844 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205, USA.
Pflugers Arch. 1998 Nov;436(6):957-61. doi: 10.1007/s004240050729.
Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the myocyte to its electrical activity via a mechanism that is not well understood. Recent pharmacological evidence suggests that KATP channels may mediate ischemic preconditioning. However, there is no potent pharmaceutical agent that specifically blocks the sarcolemmal KATP channel without significant effects on other cellular proteins. As a molecular tool, the GFG sequence in the H5 loop of the murine Kir6.2 channel was mutated to AFA. This mutated channel subunit (6.2AFA) suppressed wild-type Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, whole-cell KATP current recorded from HEK cells was greatly attenuated. The 6.2AFA subunit also co-assembled with endogenous subunits in both smooth-muscle-derived A10 cells and rat neonatal ventricular myocytes, resulting in a significant reduction of current compared with that recorded from non-transfected or mock-transfected cells (<15% of control for both cell types). This study shows that mutation of GFG-->AFA in the putative pore-forming region of Kir6.2 acts in a dominant-negative manner to suppress current in heterologous systems and in native cells.
心脏ATP敏感性钾离子（KATP）通道（SUR2A加Kir6.2）通过一种尚未完全理解的机制将心肌细胞的代谢状态与其电活动联系起来。最近的药理学证据表明，KATP通道可能介导缺血预处理。然而，目前尚无一种有效的药物能特异性阻断肌膜KATP通道而对其他细胞蛋白无显著影响。作为一种分子工具，将小鼠Kir6.2通道H5环中的GFG序列突变为AFA。这种突变的通道亚基（6.2AFA）以显性负性方式抑制野生型Kir6.2（6.2WT）通道电流：当与SUR2A和6.2WT共表达时，从HEK细胞记录到的全细胞KATP电流大大减弱。6.2AFA亚基还与平滑肌来源的A10细胞和大鼠新生心室肌细胞中的内源性亚基共同组装，与未转染或mock转染细胞记录到的电流相比，电流显著降低（两种细胞类型均<对照的15%）。本研究表明，Kir6.2假定的孔形成区域中GFG→AFA的突变以显性负性方式抑制异源系统和天然细胞中的电流。
