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 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.
Carattino
Marcelo D
MD
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, PA 15231, USA.
Sheng
Shaohu
S
Kleyman
Thomas R
TR
D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004594:Electrophysiology; D053503:Epithelial Sodium Channels; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism; D013314:Stress, Mechanical; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D018075:RNA, Complementary; D015222:Sodium Channels; D012694:Serine; D000584:Amiloride; D014364:Tryptophan; D012964:Sodium; D007930:Leucine; D003545:Cysteine; D005998:Glycine