FUNCTIONAL-CHARACTERIZATION OF A MINIMAL K+ CHANNEL EXPRESSED FROM A SYNTHETIC GENE
FUNCTIONAL-CHARACTERIZATION OF A MINIMAL K+ CHANNEL EXPRESSED FROM A SYNTHETIC GENE
复制标题
DOI：
10.1021/bi00227a025
复制
发表时间：
1991-04-02
期刊：
影响因子：
2.9
通讯作者：
MILLER, C
中科院分区：
文献类型：
--
作者：
HAUSDORFF, SF;GOLDSTEIN, SAN;MILLER, C
在已知氨基酸序列的基础上，设计并合成了一个缓慢激活的电压依赖性K+选择性离子通道基因。 将合成的基因克隆到转录载体中，并将体外转录的mRNA注射到非洲爪蟾卵母细胞中，用于产生的离子电流的电生理测定。 电流是电压依赖性的，对K+的选择性高于Na+。 一价阳离子之间的选择性遵循熟悉的K+通道顺序：K+ > Rb+ > NH 4 + > Cs+ >> Na+，Li+。 该电流被Ba 2+、Cs+和四乙基铵（TEA）抑制，它们是K+通道的常见孔阻断剂。 Cs+（而不是Ba 2+或TEA）的开放通道阻断取决于所施加的电压。 Ba 2+的主要抑制作用是通过有利于关闭状态来改变通道门控;这种作用对Ba 2+是特异性的，并且通过外部K+来缓解。 结果表明，虽然表达的多肽对于真核离子通道来说非常小，长度为130个氨基酸残基，但观察到的离子电流确实是由真正的K+通道蛋白介导的。 因此，该合成基因非常适合于K+通道功能的基本机制的分子分析。
A gene for a slowly activating, voltage-dependent K+-selective ion 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+ > Rb+ > NH4+ > Cs+ >> 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.