Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances.
Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances.
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DOI：
10.1085/jgp.200609716
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发表时间：
2007-07
影响因子：
3.8
通讯作者：
Palmer, Lawrence G
中科院分区：
文献类型：
--
作者：
Anantharam, Arun;Palmer, Lawrence G
上皮Na+通道（ENaC）是一种多聚膜蛋白，由α、β和γ三个亚基组成。每个功能通道复合物的亚基总数已被描述为遵循2α:1β:1γ的四聚体排列或3α:3β:3γ的高阶化学计量。因此，虽然很明显，所有三个ENaC亚基都需要完整的通道活动，但所需亚基的数量仍然存在争议。我们使用了一种基于非洲爪蟾卵母细胞单通道测量的新方法来解决这个问题。改变单通道电导的单个突变发生在ENaC α、β或γ亚基的孔壁残基中。从表达α、β和γ的单一物种（野生型或突变型）的卵母细胞斑块中记录的电流转变幅度具有明确的单一高斯分布。当所有三种野生型亚基的crna与等摩尔量的突变α-亚基（S589D或S592T）混合时，可以在同一斑块中观察到与纯野生型或突变型电导相对应的振幅，以及第三个中间振幅，这很可能是由至少一个野生型和至少一个突变α-亚基的通道产生的。然而，野生型和突变型βG529A或γG534E亚基的共表达没有观察到中间或杂交电导。我们的结果支持ENaC亚基的四聚体排列，其中2α， 1β和1γ聚集在中心孔周围。
The epithelial Na+ channel (ENaC) is a multimeric membrane protein consisting of three subunits, α, β, and γ. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2α:1β:1γ or a higher-ordered stoichiometry of 3α:3β:3γ. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC α, β, or γ subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of α, β, and γ showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant α-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant α-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant βG529A or γG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2α, 1β, and 1γ come together around central pore.