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A characteristic ENaC feature is sodium self-inhibition (SSI), a rapid autoregulatory mechanism in which extracellular Na\n                    <jats:sup>+<\/jats:sup>\n                    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\n                    <jats:sup>+<\/jats:sup>\n                    concentration; (2) the estimation of SSI by comparison of steady-state currents before and after chemical modification of a \u03b2\n                    <jats:sub>S520C<\/jats:sub>\n                    -ENaC mutant with MTSET; and (3) quantification of SSI from the analysis of steady-state current-voltage relationships recorded at different extracellular Na\n                    <jats:sup>+<\/jats:sup>\n                    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.\n                  <\/jats:p>","DOI":"10.1007\/s00424-026-03212-w","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T01:44:01Z","timestamp":1790559841000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Analysis of sodium self-inhibition of the epithelial sodium channel (ENaC) using classical and medium-throughput automated two-electrode voltage-clamp recordings"],"prefix":"10.1007","volume":"478","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-4486-4984","authenticated-orcid":false,"given":"Karen Lizet","family":"Luj\u00e1n L\u00f3pez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadja","family":"Furtw\u00e4ngler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carol Merlin","family":"Frank","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3554-7538","authenticated-orcid":false,"given":"Mike","family":"Althaus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1082-8656","authenticated-orcid":false,"given":"Oliver","family":"Rauh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,9,28]]},"reference":[{"key":"3212_CR1","doi-asserted-by":"publisher","unstructured":"Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, Staub O (1999) Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle\u2019s syndrome. 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