Mutations in the pore region modify epithelial sodium channel gating by shear stress
Mutations in the pore region modify epithelial sodium channel gating by shear stress
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DOI：
10.1074/jbc.m413123200
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发表时间：
2005-02-11
影响因子：
4.8
通讯作者：
Kleyman, TR
中科院分区：
文献类型：
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作者：
Carattino, MD;Sheng, SH;Kleyman, TR
先前的研究表明上皮Na+通道（ENaCs）被层流剪切应力（LSS）激活。由于突变（betaS518K）或引入的Cys残基（alphaS580C）在前秒跨膜结构域（pre-M2）的共价修饰，具有高固有开放概率的ENaCs未被LSS激活，这表明前m2区域在通道激活过程中参与了构象重排。我们通过研究野生型ENaC和Ser(576)-Ser（592）通道中Cys突变通道的LSS激活动力学，研究了α -亚基孔区在通道门化中的作用。在应用LSS之前和之后，监测表达野生型或突变型ENaCs的卵母细胞的全细胞Na+电流。延迟2.2 s后，在表达野生型ENaC的卵母细胞中观察到Na+电流单指数增加，时间常数(T)为8.1 s。C - ys在通道Ser(580) -Ser（589）的a亚基内的取代导致：(i)在LSS激活通道之前延迟时间减少(Ser(580) -Trp（585), Gly(587)）或增加（Ser(589)）， （ii）通道激活速率增加(Gln(581), Leu(584), Trp(585), Phe（586) Ser(588)）或减少（Ser(189)），或（iii）响应强度降低(Ser(583), Gly（587), Leu(584)）。在假定的酰胺结合位点（alphaSer（583）或betaGly(525)）或选择性过滤器（alphaGly(587)）中替换Cys导致LSS响应降低，并表现出多指数的激活时间过程。相应的γ亚单位突变体（alphabetagammaG542C）对LSS的响应最小，并表现出高的内在开放概率。这些数据表明，孔区的残留物参与了导致通道激活的机械刺激的感知和/或转导，这与ENaC孔区在调节通道门控中起关键作用的假设是一致的。
Previous studies have shown that epithelial Na+ channels (ENaCs) are activated by laminar shear stress (LSS). ENaCs with a high intrinsic open probability because of a mutation betaS518K) or covalent modification of an introduced Cys residue (alphaS580C) in the pre-second transmembrane domain (pre-M2) were not activated by LSS, suggesting that the pre-M2 region participates in conformational rearrangements during channel activation. We examined the role of the pore region of the alpha-subunit in channel gating by studying the kinetics of activation by LSS of wild-type ENaC and channels with Cys mutations in the tract Ser (576)-Ser(592). Whole cell Na+ currents were monitored in oocytes expressing wildtype or mutant ENaCs prior to and following application of LSS. Following a 2.2-s delay, a monoexponential increase in Na+ currents was observed with a time constant (T) of 8.1 s in oocytes expressing wild-type ENaC. C ys substitutions within the a-subunit in the tract Ser (580)-Ser(589) resulted in: (i) a reduction (Ser(580) -Trp(585), Gly(587)) or increase (Ser(589)) in delay times preceding channel activation by LSS, (ii) an increase (Gln(581), Leu(584), Trp(585), Phe(586) Ser(588)) or decrease (Ser(189)) in the rate of channel activation, or (iii) a decrease in the magnitude of the response (Ser(583), Gly(587), Leu(584)). Cys substitutions at a putative amiloride-binding site (alphaSer(583) or betaGly(525)) or within the selectivity filter (alphaGly(587)) resulted in a reduction in the LSS response, and exhibited a multiexponential time course of activation. The corresponding gamma-subunit mutant (alphabetagammaG542C had a minimal response to LSS and exhibited a high intrinsic open probability. These data suggest that residues in the pore region participate in the sensing and/or transduction of the mechanical stimulus that results in channel activation and are consistent with the hypothesis that the ENaC pore region has a key role in modulating channel gating.