Tzounopoulos T, Maylie J, Adelman J P
Vollum Institute, Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland 97201, USA.
Biophys J. 1995 Sep;69(3):904-8. doi: 10.1016/S0006-3495(95)79964-7.
Xenopus oocytes are widely employed for heterologous expression of cloned proteins, particularly electrogenic molecules such as ion channels and transporters. The high levels of expression readily obtained permit detailed investigations without interference from endogenous conductances. Injection of min K mRNA into Xenopus oocytes results in expression of voltage-dependent potassium-selective channels. Recent data show that injections of high concentrations of min K mRNA also induce a chloride current with very different biophysical, pharmacological, and regulatory properties from the min K potassium current. This led to the suggestion that the min K protein acts as an inducer of endogenous, normally silent oocyte ion channels. We now report that high levels of heterologous expression of many membrane proteins in Xenopus oocytes specifically induce this chloride current and a hyperpolarization-activated cation-selective current. The current is blocked by 4,4'-diisothiocyanostilbene-2-2'-disulphonic acid and tetraethylammonium, enhanced by clofilium, and is pH-sensitive. Criteria are presented that distinguish this endogenous current from those due to heterologous expression of electrogenic proteins in Xenopus oocytes. Together with structure-function studies, these results support the hypothesis that the min K protein comprises a potassium-selective channel.
非洲爪蟾卵母细胞被广泛用于克隆蛋白的异源表达，尤其是离子通道和转运体等生电分子。其易于获得的高表达水平使得能够在不受内源性电导干扰的情况下进行详细研究。将min K信使核糖核酸注射到非洲爪蟾卵母细胞中会导致电压依赖性钾选择性通道的表达。最近的数据表明，注射高浓度的min K信使核糖核酸还会诱导出一种氯离子电流，其生物物理、药理和调节特性与min K钾电流非常不同。这引发了一种观点，即min K蛋白作为内源性、通常沉默的卵母细胞离子通道的诱导剂。我们现在报告，非洲爪蟾卵母细胞中许多膜蛋白的高水平异源表达会特异性地诱导这种氯离子电流和一种超极化激活的阳离子选择性电流。该电流被4,4'-二异硫氰基芪-2,2'-二磺酸和四乙铵阻断，被氯非铵增强，并且对pH敏感。本文提出了将这种内源性电流与非洲爪蟾卵母细胞中生电蛋白异源表达所产生的电流区分开来的标准。结合结构-功能研究，这些结果支持了min K蛋白构成钾选择性通道的假说。
