Analysis of sodium self-inhibition of the epithelial sodium channel (ENaC) using classical and medium-throughput automated two-electrode voltage-clamp recordings

The epithelial sodium channel (ENaC) is a key regulator of electrolyte homeostasis and blood pressure. A characteristic ENaC feature is sodium self-inhibition (SSI), a rapid autoregulatory mechanism in which extracellular Na + ions allosterically bind ENaC and facilitate transition into a low open probability state. Although SSI is an important functional parameter for assessing ENaC activity and gating, its quantification has traditionally relied on manual two-electrode voltage-clamp (TEVC) recordings with limited scalability and throughput. Here, we improved and evaluated experimental strategies and analytical methods for robust quantification of ENaC SSI in manual and automated TEVC systems: (1) The determination of SSI from extrapolation of transient current recordings in response to a jump in the extracellular Na + concentration; (2) the estimation of SSI by comparison of steady-state currents before and after chemical modification of a β S520C -ENaC mutant with MTSET; and (3) quantification of SSI from the analysis of steady-state current-voltage relationships recorded at different extracellular Na + concentrations. The latter procedure is the most reliable for SSI quantification in perfusion rate-limited automated TEVC systems. These methods can expand the capabilities of automated electrophysiology and support medium- to high-throughput investigation of autoregulatory mechanisms in ENaC variants and the discovery of novel ENaC modulators.