Carattino Marcelo D, Sheng Shaohu, Kleyman Thomas R
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, PA 15231, USA.
J Biol Chem. 2005 Feb 11;280(6):4393-401. doi: 10.1074/jbc.M413123200. Epub 2004 Nov 29.
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 Ser576-Ser592. Whole cell Na+ currents were monitored in oocytes expressing wild-type 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 (tau) of 8.1 s in oocytes expressing wild-type ENaC. Cys substitutions within the alpha-subunit in the tract Ser580-Ser589 resulted in: (i) a reduction (Ser580-Trp585, Gly587) or increase (Ser589) in delay times preceding channel activation by LSS, (ii) an increase (Gln581, Leu584, Trp585, Phe586, Ser588) or decrease (Ser589) in the rate of channel activation, or (iii) a decrease in the magnitude of the response (Ser583, Gly587, Leu584). Cys substitutions at a putative amiloride-binding site (alphaSer583 or betaGly525) or within the selectivity filter (alphaGly587) 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.
先前的研究表明，上皮钠通道（ENaC）可被层流切应力（LSS）激活。由于在第二个跨膜结构域前体（pre-M2）中发生突变（βS518K）或对引入的半胱氨酸残基（αS580C）进行共价修饰而具有高固有开放概率的ENaC未被LSS激活，这表明pre-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和在Ser576-Ser592区域具有半胱氨酸突变的通道对LSS激活的动力学，来研究α亚基孔区在通道门控中的作用。在施加LSS之前和之后，监测表达野生型或突变型ENaC的卵母细胞中的全细胞钠电流。经过2.2秒的延迟后，在表达野生型ENaC的卵母细胞中观察到钠电流呈单指数增加，时间常数（τ）为8.1秒。α亚基中Ser580-Ser589区域内的半胱氨酸替代导致：（i）LSS激活通道之前的延迟时间减少（Ser580-Trp585、Gly587）或增加（Ser589），（ii）通道激活速率增加（Gln581、Leu584、Trp585、Phe586、Ser588）或降低（Ser589），或（iii）反应幅度降低（Ser583、Gly587、Leu584）。在假定的氨氯地平结合位点（αSer583或βGly525）或选择性过滤器内（αGly587）的半胱氨酸替代导致LSS反应降低，并呈现多指数激活时间进程。相应的γ亚基突变体（αβγG542C）对LSS的反应最小，且具有高固有开放概率。这些数据表明，孔区中的残基参与了导致通道激活的机械刺激的感知和/或转导，并且与ENaC孔区在调节通道门控中起关键作用的假设一致。
