Abstract
The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.
Highlights
The highly selective and amiloride-sensitive epithelial sodium channel ENaC1 expressed at the apical membrane of tight epithelia represents the main pathway for sodium absorption in the aldosterone-sensitive distal nephron and the colon
We have shown for the first time that ENaC can be blocked from the cytosolic side by sulfhydryl-reactive MTS reagents and by the transition metal cations Cd2ϩ and Zn2ϩ reacting with thiol groups of cysteine residues
No significant changes in single channel current amplitude were observed in the presence of the intracellular thiol reagents
Summary
Lular cysteine residues in ENaC is unknown, and so far, no study has been conducted to determine the effects of chemical modifications or substitutions of these intracellular cysteine residues on ENaC function. We found that ENaC is highly sensitive to intracellular thiol reagents and thiol oxidation, suggesting a possible adaptation of ENaC activity to changes in the intracellular redox potential. Multiple cysteine residues located in the vicinity of an intracellular gating domain in the amino-terminal region of ENaC subunits as well as in the carboxyl termini of ␣ and  ENaC subunits are involved in ENaC inhibition mediated by intracellular thiol reagents
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