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J. Biol. Chem., Vol. 276, Issue 47, 44091-44098, November 23, 2001

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Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity^*

Shaohu Sheng^§, Kathleen A. McNulty^¶, Johanna M. Harvey^¶, and Thomas R. Kleyman^**

From the Departments of  Medicine and of  Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the ^¶ Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (, , and ). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human -ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse ENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (Glu⁵⁹⁵, Glu⁵⁹⁸, and Asp⁶⁰²) led to a significant reduction of expressed amiloride-sensitive Na⁺ currents. Two mutations (E595C and D602C) resulted in K⁺-permeable channels whereas multiple mutations altered Li⁺/Na⁺ current ratios. Channels containing D602K or D602A also conducted K⁺ whereas more conservative mutations (D602E and D602N) retained wild type selectivity. Cysteine substitution at the site equivalent to Asp⁶⁰² within  mENaC (D544C) did not alter either Li⁺/Na⁺ or K⁺/Na⁺ current ratios, although mutation of the equivalent site within  mENaC (D562C) significantly increased the Li⁺/Na⁺ current ratio. Mutants containing introduced cysteine residues at Glu⁵⁹⁵, Glu⁵⁹⁸, Asp⁶⁰², or Thr⁶⁰⁷ did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of ENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (Glu⁵⁹⁵ and Asp⁶⁰²) may have a role in conferring ion selectivity.

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