Po S, Snyders D J, Baker R, Tamkun M M, Bennett P B
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN.
Circ Res. 1992 Sep;71(3):732-6. doi: 10.1161/01.res.71.3.732.
Recently a putative K+ channel with homology to the Shaker family of potassium channels has been cloned from human ventricular myocardium. However, proof that the cDNA encodes a K+ channel requires appropriate translation and expression of a functional ion-selective channel. Therefore, expression of this putative human K+ channel DNA was attempted by cytoplasmic injections of in vitro transcribed cRNA into Xenopus laevis oocytes and screening by two-electrode voltage-clamp methods. This resulted in expression of voltage-gated channels that rapidly inactivated (time constant of inactivation, 47.6 +/- 3.6 msec; 0 mV; n = 10) and were at least 50 times more selective for K+ than Na+ (Na+/K+ permeability ratio of 0.02). The channels showed voltage-dependent activation (half-maximal voltage, -34 +/- 0.7 mV; n = 5), and 50% of the channels were inactivated within 2 seconds when the membrane potential was clamped near -60 mV (half-maximal voltage, -62 +/- 7 mV; n = 10). The expressed protein resulted in a K+ current that had many properties similar to the 4-aminopyridine-sensitive calcium-insensitive component of the cardiac transient outward current that is observed in native cardiac myocytes and thus may serve as one molecular substrate for this current.
最近，已从人心室心肌中克隆出一种与钾通道的Shaker家族具有同源性的假定钾通道。然而，要证明该cDNA编码钾通道，需要对功能性离子选择性通道进行适当的翻译和表达。因此，通过将体外转录的cRNA胞质注射到非洲爪蟾卵母细胞中，并采用双电极电压钳法进行筛选，来尝试表达这种假定的人钾通道DNA。这导致了电压门控通道的表达，这些通道迅速失活（失活时间常数为47.6±3.6毫秒；0 mV；n = 10），并且对K+的选择性比对Na+至少高50倍（Na+/K+通透性比率为0.02）。这些通道表现出电压依赖性激活（半数最大激活电压为-34±0.7 mV；n = 5），当膜电位钳制在-60 mV附近时，50%的通道在2秒内失活（半数最大失活电压为-62±7 mV；n = 10）。所表达的蛋白质产生了一种钾电流，其许多特性与在天然心肌细胞中观察到的心脏瞬时外向电流的4-氨基吡啶敏感、钙不敏感成分相似，因此可能是该电流的一种分子底物。
