{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T06:48:37Z","timestamp":1790318917274,"version":"4.1.0"},"reference-count":25,"publisher":"Rockefeller University Press","issue":"5","content-domain":{"domain":["rupress.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[1998,11,1]]},"abstract":"<jats:p>The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human\u2013shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2\u20137 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the Ki(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 Kis.<\/jats:p>","DOI":"10.1085\/jgp.112.5.549","type":"journal-article","created":{"date-parts":[[2002,7,26]],"date-time":"2002-07-26T16:50:20Z","timestamp":1027702220000},"page":"549-558","update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":84,"title":["Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding"],"prefix":"10.1085","volume":"112","author":[{"given":"Paul","family":"Isenring","sequence":"first","affiliation":[{"name":"From the Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven C.","family":"Jacoby","sequence":"additional","affiliation":[{"name":"From the Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Chang","sequence":"additional","affiliation":[{"name":"From the Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"Bliss","family":"Forbush","sequence":"additional","affiliation":[{"name":"From the Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[1998,11,1]]},"reference":[{"key":"2023072918131666900_B1","doi-asserted-by":"crossref","first-page":"22764","DOI":"10.1074\/jbc.270.39.22764","article-title":"Substitutions of serine 775 in the alpha subunit of the Na,K-ATPase selectively disrupt K+ high affinity activation without affecting Na+interaction","volume":"270","author":"Arguello","year":"1995","journal-title":"J Biol Chem"},{"key":"2023072918131666900_B2","first-page":"F473","article-title":"A quantitative description of the Na,K,2Cl cotransporter and its conformity with experimental data","volume":"273","author":"Benjamin","year":"1997","journal-title":"Am J Physiol"},{"key":"2023072918131666900_B3","doi-asserted-by":"crossref","first-page":"25677","DOI":"10.1016\/S0021-9258(18)47302-4","article-title":"Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl\u2212cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells","volume":"269","author":"Delpire","year":"1994","journal-title":"J Biol Chem"},{"key":"2023072918131666900_B4","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1126\/science.280.5360.69","article-title":"The structure of the potassium channel: molecular basis of K+conduction and selectivity","volume":"280","author":"Doyle","year":"1998","journal-title":"Science"},{"key":"2023072918131666900_B5","doi-asserted-by":"crossref","first-page":"11787","DOI":"10.1016\/S0021-9258(17)44299-2","article-title":"3H-Bumetanide binding to membranes isolated from dog kidney outer medulla. 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