Kosari Farhad, Sheng Shaohu, Kleyman Thomas R
Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA.
Methods Mol Biol. 2006;337:53-63. doi: 10.1385/1-59745-095-2:53.
The amiloride-sensitive epithelial Na+ channel (ENaC) is typically composed of three structurally related subunits termed alpha, beta, and gamma. We describe methods to determine the functional subunit stoichiometry of ENaC based on a biophysical approach that was first introduced in 1991 to determine the subunit stoichiometry of a voltage-gated K+ channel. The strategy is to analyze channel sensitivity to a specific blocker when various mixtures of block-sensitive and blocker-insensitive subunits are coexpressed in a heterologous expression system. Details related to the expression of wild type and mutant ENaCs in Xenopus oocytes and the examination of blocker sensitivity by two-electrode voltage clamp, as well as analysis of data are provided.
氨氯地平敏感的上皮钠离子通道（ENaC）通常由三个结构相关的亚基组成，分别称为α、β和γ。我们描述了基于一种生物物理方法来确定ENaC功能亚基化学计量比的方法，该方法于1991年首次引入，用于确定电压门控钾通道的亚基化学计量比。该策略是当对阻滞剂敏感和不敏感的亚基的各种混合物在异源表达系统中共表达时，分析通道对特定阻滞剂的敏感性。文中提供了与野生型和突变型ENaC在非洲爪蟾卵母细胞中的表达、通过双电极电压钳检测阻滞剂敏感性以及数据分析相关的详细信息。
