Asymmetric Organization of the Pore Region of the Epithelial Sodium Channel

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the β-subunit that line the pore, we systematically mutated residues Gln523–Ile536 to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li+ to Na+ current ratios, whereas only cysteine replacement of βGly529resulted in K+-permeable channels. Besides βG525C, large increases in the inhibitory constant of amiloride were observed with mutations at βGly529 and βSer531 within the previously identified 3-residue tract that restricts K+permeation. Cysteine substitution preceding (βPhe524 and βGly525), within (βGly530) or following (βLeu533) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at βGln523, βPhe524, and βTrp527. MTSET did not inhibit αβG525Cγ, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the α- and γ-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.