Raja Mobeen, Vales Elisabeth
Institute for Biophysics, Johannes Kepler University Linz, 4040 Linz, Austria.
Biophys Chem. 2009 Oct;144(3):95-100. doi: 10.1016/j.bpc.2009.06.006. Epub 2009 Jun 27.
All K(+)-channels are stabilized by K(+)-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K(+)-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K(+)-channel.
所有钾离子通道在选择性过滤器中都由钾离子稳定。然而，它们在选择性过滤器方面彼此不同。在本研究中，我们将KcsA选择性过滤器中的特定残基缬氨酸-76替换为内向整流钾通道（Kir）中的对应异亮氨酸。通过传统凝胶电泳测定，四聚体完全转化为单体。然而，通过全氟辛酸（PFO）凝胶电泳，突变通道大多被检测为四聚体。色氨酸荧光和丙烯酰胺猝灭实验表明，通过局部环境亲水性的增加，通道折叠特性发生了显著改变。此外，在平面脂质双层实验中，与未修饰的KcsA相比，V76I表现出极低的电导率和缩短的通道开放时间。这些研究表明，V76I可能有助于确定选择性钾通道中的稳定、折叠和通道门控特性。
