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J. Biol. Chem., Vol. 280, Issue 6, 4393-4401, February 11, 2005

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Mutations in the Pore Region Modify Epithelial Sodium Channel Gating by Shear Stress^*

Marcelo D. Carattino, Shaohu Sheng, and Thomas R. Kleyman¶||

From the Renal-Electrolyte Division, Department of Medicine and the ^¶Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

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 (S518K) or covalent modification of an introduced Cys residue (S580C) 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 -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 () of 8.1 s in oocytes expressing wild-type ENaC. Cys substitutions within the -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 (Ser⁵⁸³ or Gly⁵²⁵) or within the selectivity filter (Gly⁵⁸⁷) resulted in a reduction in the LSS response, and exhibited a multiexponential time course of activation. The corresponding -subunit mutant (G542C) 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.

Received for publication, November 19, 2004

^* This work was supported by Grants DK51391 and DK38470 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a postdoctoral fellowship award from the Pennsylvania-Delaware Affiliate of the American Heart Association.

^|| To whom correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-646-3121; Fax: 412-648-9166; E-mail: kleyman{at}pitt.edu.

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