Hausdorff S F, Goldstein S A, Rushin E E, Miller C
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254-9110.
Biochemistry. 1991 Apr 2;30(13):3341-6. doi: 10.1021/bi00227a025.
A gene for a slowly activating, voltage-dependent K(+) -selective channel was designed and synthesized on the basis of its known amino acid sequence. The synthetic gene was cloned into a transcription vector, and in vitro transcribed mRNA was injected into Xenopus oocytes for electrophysiological assay of the resulting ionic currents. The currents are voltage-dependent and highly selective for K+ over Na+. The selectivity among monovalent cations follows a familiar K(+)- channel sequence: K+ greater than Rb+ greater than NH4+ greater than Cs+ much greater than Na+, Li+. The currents are inhibited by Ba2+, Cs+, and tetraethylammonium (TEA), common pore blockers of K+ channels. Open-channel blockade by Cs+ (but not by Ba2+ or TEA) depends on applied voltage. The major inhibitory effect of Ba2+ is to alter channel gating by favoring the closed state; this effect is specific for Ba2+ and is relieved by external K+. The results argue that although the polypeptide expressed is very small for a eukaryotic ion channel, 130 amino acid residues in length, the ionic currents observed are indeed mediated by a genuine K(+) -channel protein. This synthetic gene is therefore well suited for a molecular analysis of the basic mechanisms of K(+) -channel function.
基于已知的氨基酸序列设计并合成了一个编码缓慢激活、电压依赖性钾离子选择性通道的基因。将合成基因克隆到转录载体中，体外转录的mRNA被注入非洲爪蟾卵母细胞中，用于对产生的离子电流进行电生理分析。这些电流具有电压依赖性，对钾离子的选择性远高于钠离子。单价阳离子之间的选择性遵循常见的钾离子通道序列：钾离子＞铷离子＞铵离子＞铯离子＞＞钠离子、锂离子。这些电流受到钡离子、铯离子和四乙铵（TEA）的抑制，它们是钾离子通道常见的孔道阻断剂。铯离子（但不是钡离子或TEA）对开放通道的阻断取决于施加的电压。钡离子的主要抑制作用是通过促进通道处于关闭状态来改变通道门控；这种作用对钡离子具有特异性，并且可被细胞外钾离子缓解。结果表明，尽管所表达的多肽对于真核离子通道来说非常小，仅130个氨基酸残基，但观察到的离子电流确实是由真正的钾离子通道蛋白介导的。因此，这个合成基因非常适合对钾离子通道功能的基本机制进行分子分析。
