Abstract
The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).
Highlights
Grants R01 DK054354, R01 ES014701, and P30 DK079307. 1 To whom correspondence should be addressed: Renal-Electrolyte Division, mechanism for epithelial cells to rapidly tune the rate of Naϩ influx according to fluctuations of the urinary [Naϩ]
A functional epithelial Na؉ channel (ENaC) complex likely is formed by three homologous subunits in a manner similar to the trimeric structure of chicken acid-sensing ion channel 1 [3], a member of the ENaC/degenerin superfamily
To gain additional insights regarding the structural requirements of Naϩ self-inhibition, we systematically investigated the functional roles of selected residues in the vicinity of ␥His239, which we previously termed the extracellular allosteric regulatory site (EARS) [5, 8]
Summary
Site-directed Mutagenesis—Point mutations were introduced into mouse ␣, , and ␥ ENaC (␣␥mENaC) cDNAs in pBluescript SK-vector (Stratagene, La Jolla, CA) using the QuikChange II XL site-directed mutagenesis kit (Stratagene). ENaC Expression and Two-electrode Voltage Clamp—ENaC expression in Xenopus oocytes and current measurements by two-electrode voltage clamp were performed as reported previously [15]. Voltage clamp was performed using a TEV-200 voltage clamp amplifier (Dagan Corp.) and the DigiData 1322A interface controlled by pClamp (version 9.2, Molecular Devices Corp., Sunnyvale, CA). Upon completion of the experiment, 10 M amiloride was added to the bath solution to determine the amiloride-insensitive portion of the whole cell current. An exponential equation by Clampfit (version 9.2, Molecular Devices Corp.) was used to fit the first 40 s of current decay following the maximal inward current (peak current, Ipeak) directly after the bath solution change from a low to high Naϩ concentration. A p value of Ͻ0.05 was considered significantly different
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