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J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Serious neurological disorders are caused by mutation of Na+,K+-ATPase \u03b11\u2013\u03b13 isoforms. Several disease mutations reduce the affinity for Na+, which in vivo results in an increase in the intracellular Na+ concentration with a secondary rise in intracellular Ca2+. Current treatments are ineffective, as they do not address Na+,K+-ATPase function. Hence, a platform for the development of a rational therapeutic approach that restores Na+ affinity is needed. Using the COS-1 cell expression system, one of the most conspicuous reductions in Na+ affinity of Na+,K+-ATPase has been reported for the neurological disease mutation \u03b13-D923N of the \u03b13 isoform. We describe here second-site mutations (\u201crescue mutations\u201d) that fully restore Na+ affinity of \u03b13-D923N and the corresponding rat \u03b11-D928N and reveal the underlying molecular mechanism. Paradoxically, this insight evolved from investigation of a rapid-onset ataxia Na+,K+-ATPase mutant, \u03b13-G316S, that exhibits an increased affinity for Na+. When applied as second-site mutations, \u03b13-G316S and the corresponding rat \u03b11-G321S are super-efficient rescuers of the reduced Na+ affinity of \u03b13-D923N and rat \u03b11-D928N. Replacement of the glycine with serine leads to a clash between transmembrane helices in the K+-bound E2 state of Na+,K+-ATPase but not in the Na+-bound E1 state. A similarly strong rescuing power is observed for second-site mutations breaking a salt bridge present only in the E2 state. The rescue is due to a structural change resembling the rearrangement of the ion binding sites occurring during the normal Na+,K+-ATPase transport cycle as part of the E2-to-E1 conformational transition.<\/jats:p>","DOI":"10.3390\/ijms27188424","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T13:32:12Z","timestamp":1790083932000},"page":"8424","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mechanism of Rescue of Na+,K+-ATPase Function in Neurological Disease Revealed by New Super-Efficient Second-Site Mutations"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3931-4640","authenticated-orcid":false,"given":"Rikke","family":"Holm","sequence":"first","affiliation":[{"name":"Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2064-5861","authenticated-orcid":false,"given":"Mads S.","family":"Toustrup-Jensen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Hang N.","family":"Nielsen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0654-4300","authenticated-orcid":false,"given":"Jens Peter","family":"Andersen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4727-9382","authenticated-orcid":false,"given":"Bente","family":"Vilsen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1016\/0006-3002(57)90343-8","article-title":"The influence of some cations on an adenosine triphosphatase from peripheral nerves","volume":"23","author":"Skou","year":"1957","journal-title":"Biochim. 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