Role of the Wrist Domain in the Response of the Epithelial Sodium Channel to External Stimuli

The epithelial Na+ channel (ENaC) is regulated by a variety of external factors that alter channel activity by inducing conformational changes within its large extracellular region that are transmitted to the gate. The wrist domain consists of small linkers connecting the extracellular region to the transmembrane domains, where the channel pore and gate reside. We employed site-directed mutagenesis combined with two-electrode voltage clamp to investigate the role of the wrist domain in channel gating in response to extracellular factors. Channel inhibition by external Na+ was reduced by selected mutations within the wrist domain of the alpha subunit, likely reflecting an increase in channel open probability. The most robust changes were observed when Cys was introduced at alpha Pro-138 and alpha Ser-568, sites immediately adjacent to the palm domain. In addition, one of these Cys mutants exhibited an enhanced response to shear stress. In the context of channels that have a low open probability due to retention of an inhibitory tract, the response to external Na+ was reduced by Cys substitutions at both alpha Pro-138 and alpha Ser-568. We observed a significant correlation between changes in channel inhibition by external Na+ and the relative response to shear stress for the alpha subunit mutants that were examined. Mutants that exhibited reduced inhibition by external Na+ also showed an enhanced response to shear stress. Together, our data suggest that the wrist domain has a role in modulating the channel's response to external stimuli.