Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

Abstract

The epithelial Na ؉ channel (ENaC) is typically formed by three homologous subunits (␣, ␤, and ␥) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na ؉ self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na ؉ . All 16 Cys residues within ␣ and ␥ ECLs and selected ␤ ECL Cys residues were individually mutated to alanine or serine residues. The Na ؉ self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight ␣ (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one ␤ (Cys-7), and nine ␥ (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na ؉ self-inhibition. Na ؉ self-inhibition was eliminated by simultaneous mutations of either the last three ␣ ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both ␣ and ␥ ECLs. By analyzing the Na ؉ self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the ␣ECL (␣Cys-1/␣Cys-6, ␣Cys-4/␣Cys-5, ␣Cys-7/␣Cys-16, ␣Cys-10/␣Cys-13, and ␣Cys-11/␣Cys-12) and one pair within the ␥ECL (␥Cys-7/␥Cys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the ␣ and ␥ ENaC subunits are required to establish the tertiary structure that ensures a proper Na ؉ self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.

Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains

“…Given the similarities in amino acid sequences and biophysical properties among ASIC and other members of ENaC/degenerin family, it is very likely that ENaC subunits adopt a similar overall tertiary structure, although considerable differences likely exist within local structures, particularly for poorly conserved regions that likely contribute to differences in the biophysical properties of ASIC and ENaC. Additional evidence supporting the notion that ECDs of ASIC and ENaC share a similar overall structure is that the disulfide bridges proposed for the ECD of ␣ ENaC, based on mutational analyses and chemical modifications, match the disulfide bonds within the cASIC1 structure (9,14).…”

“…We recently reported that point mutations at multiple ␣ and ␥ ECD Cys residues blunted Na ϩ self-inhibition, and certain double or triple mutations rendered ENaC insensitive to high concentration of extracellular Na ϩ . These results suggest that multiple Cys residues are required to establish the proper tertiary structure permitting this allosteric regulation (9). In addition, the N-terminal portion of ECD contains ␥His 239 , a previously identified residue critical for Na ϩ self-inhibition, as well as defined protease cleavage sites (4, 10 -12).…”

“…Membrane-impermeant and positively changed MTSET was chosen to avoid modifications of intracellular sulfhydryl groups (25) and because it does not significantly alter the current of WT ␣␤␥ mENaC (9). MTSET (1 mM) applied in the bath solution slowly inhibited currents in oocytes expressing ␣␤␥M438C mENaC with a maximal inhibition of 56 Ϯ 2% (n ϭ 6) within 2 min and a time constant of 40 Ϯ 2 s (n ϭ 6; Fig.…”

“…The ␥Met 438 is near the 13th conserved Cys (␥Cys 440 ) whose mutation leads to significant suppression of Na ϩ selfinhibition (9). Sequence alignments placed this residue at a site corresponding to Lys 342 within cASIC1, which is located at the N-terminal part of helix ␣5, one of the two helices that comprise the thumb domain (Fig.…”

Inhibitory Tract Traps the Epithelial Na+ Channel in a Low Activity Conformation

“…The thumb domains of the ENaC/degenerin family of ion channels are poorly conserved yet share two important features: two predicted helices and 10 absolutely conserved Cys that form a five-disulfide bond ladder (12,15). These hallmark features of ENaC/degenerin channels suggest that the antiparallel arrangement of the two thumb helices in ASIC1 is widely conserved.…”