Xu Xianghua, Kanda Vikram A, Choi Eun, Panaghie Gianina, Roepke Torsten K, Gaeta Stephen A, Christini David J, Lerner Daniel J, Abbott Geoffrey W
Greenberg Division of Cardiology, Department of Medicine, Weill Medical College of Cornell University, Starr 463, 520 East 70th Street, New York, NY 10065, USA.
Cardiovasc Res. 2009 Jun 1;82(3):430-8. doi: 10.1093/cvr/cvp047. Epub 2009 Feb 7.
KCNQ1-MinK potassium channel complexes (4alpha:2beta stoichiometry) generate IKs, the slowly activating human cardiac ventricular repolarization current. The MinK ancillary subunit slows KCNQ1 activation, eliminates its inactivation, and increases its unitary conductance. However, KCNQ1 transcripts outnumber MinK transcripts five to one in human ventricles, suggesting KCNQ1 also forms other heteromeric or even homomeric channels there. Mechanisms governing which channel types prevail have not previously been reported, despite their significance: normal cardiac rhythm requires tight control of IKs density and kinetics, and inherited mutations in KCNQ1 and MinK can cause ventricular fibrillation and sudden death. Here, we describe a novel mechanism for this control.
Whole-cell patch-clamping, confocal immunofluorescence microscopy, antibody feeding, biotin feeding, fluorescent transferrin feeding, and protein biochemistry techniques were applied to COS-7 cells heterologously expressing KCNQ1 with wild-type or mutant MinK and dynamin 2 and to native IKs channels in guinea-pig myocytes. KCNQ1-MinK complexes, but not homomeric KCNQ1 channels, were found to undergo clathrin- and dynamin 2-dependent internalization (DDI). Three sites on the MinK intracellular C-terminus were, in concert, necessary and sufficient for DDI. Gating kinetics and sensitivity to XE991 indicated that DDI decreased cell-surface KCNQ1-MinK channels relative to homomeric KCNQ1, decreasing whole-cell current but increasing net activation rate; inhibiting DDI did the reverse.
The data redefine MinK as an endocytic chaperone for KCNQ1 and present a dynamic mechanism for controlling net surface Kv channel subunit composition-and thus current density and gating kinetics-that may also apply to other alpha-beta type Kv channel complexes.
KCNQ1 - MinK钾通道复合物（化学计量比为4α:2β）产生IKs，即缓慢激活的人类心室复极电流。MinK辅助亚基减缓KCNQ1的激活，消除其失活，并增加其单位电导。然而，在人类心室中，KCNQ1转录本数量比MinK转录本多五倍，这表明KCNQ1在那里也形成其他异源或甚至同源通道。尽管其具有重要意义，但此前尚未报道决定哪种通道类型占主导的机制：正常心律需要严格控制IKs密度和动力学，而KCNQ1和MinK的遗传突变可导致心室颤动和猝死。在此，我们描述了一种新的控制机制。
全细胞膜片钳、共聚焦免疫荧光显微镜、抗体导入、生物素导入、荧光转铁蛋白导入和蛋白质生物化学技术应用于异源表达野生型或突变型MinK以及发动蛋白2的COS - 7细胞，以及豚鼠心肌细胞中的天然IKs通道。发现KCNQ1 - MinK复合物而非同源KCNQ1通道经历网格蛋白和发动蛋白2依赖性内吞作用（DDI）。MinK细胞内C末端的三个位点共同对DDI是必需且充分的。门控动力学和对XE991的敏感性表明，相对于同源KCNQ1，DDI降低了细胞表面的KCNQ1 - MinK通道，减少了全细胞电流但增加了净激活率；抑制DDI则产生相反效果。
这些数据将MinK重新定义为KCNQ1的内吞伴侣，并提出了一种控制净表面Kv通道亚基组成以及电流密度和门控动力学的动态机制，该机制可能也适用于其他α - β型Kv通道复合物。
