Potet Franck, Petersen Christina I, Boutaud Olivier, Shuai Wen, Stepanovic Svetlana Z, Balser Jeffrey R, Kupershmidt Sabina
Department of Anesthesiology, Vanderbilt University, Nashville, TN 37232, USA.
J Mol Cell Cardiol. 2009 Feb;46(2):257-67. doi: 10.1016/j.yjmcc.2008.10.015. Epub 2008 Nov 5.
The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of I(Kr), a K(+) current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that I(Kr) recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53% enhancement of I(Kr). ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG.
人类醚 - 去极化相关基因（HERG）构成了I(Kr)的孔形成亚基，I(Kr)是一种参与心脏动作电位复极化的钾离子电流。虽然HERG突变使患者易患心律失常（长QT综合征；LQTS），但HERG调节因子功能的改变无疑也是LQTS的危险因素。我们将RNA干扰与秀丽隐杆线虫的行为筛选相结合，以检测影响HERG同源物UNC - 103功能的基因。其中一个这样的基因编码rho - GTPase激活蛋白6（ARHGAP6）的线虫直系同源物。除了其GAP功能外，ARHGAP6还诱导细胞骨架重排并激活磷脂酶C（PLC）。在这里我们表明，与单独表达HERG相比，共表达HERG和ARHGAP6的细胞中记录的I(Kr)降低了43%。对细胞表面相关HERG的生化测量显示，ARHGAP6使HERG的膜表达降低了35%，这与电流的降低密切相关。在心房肌细胞系中，通过病毒转导的短发夹RNA抑制内源性ARHGAP6导致I(Kr)增强了53%。当我们引入显性负性rho - GTPase或缺乏rhoGAP功能的ARHGAP6时，ARHGAP6的作用得以维持，表明ARHGAP6对HERG的调节独立于rho激活。然而，当PLC被抑制时，ARHGAP6失去了效力。我们进一步确定，ARHGAP6的作用是由HERG C末端的一个共有SH3结合结构域介导的，尽管未观察到稳定的ARHGAP6 - HERG复合物。这些数据将rhoGAP激活的PLC途径与HERG膜表达联系起来，并暗示这类蛋白质家族是涉及HERG的疾病中的候选基因。
