Mutations in the pore region modify epithelial sodium channel gating by shear stress

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.