{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T13:07:07Z","timestamp":1769519227495,"version":"3.49.0"},"reference-count":45,"publisher":"Wiley","issue":"16","license":[{"start":{"date-parts":[[2017,6,27]],"date-time":"2017-06-27T00:00:00Z","timestamp":1498521600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100004663","name":"Ministry of Science and Technology, Taiwan","doi-asserted-by":"publisher","award":["103\u20102628\u2010B\u2010016\u2010001\u2010MY3"],"award-info":[{"award-number":["103\u20102628\u2010B\u2010016\u2010001\u2010MY3"]}],"id":[{"id":"10.13039\/501100004663","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["DK109887"],"award-info":[{"award-number":["DK109887"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["The Journal of Physiology"],"published-print":{"date-parts":[[2017,8,15]]},"abstract":"<jats:sec><jats:title>Key points<\/jats:title><jats:p><jats:list list-type=\"bullet\">\n<jats:list-item><jats:p>The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of <jats:italic>CLCNKB<\/jats:italic> mutations may contribute to the phenotypic variability, and the genotype\u2013phenotype association has not been established.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>Low\u2010level expression of the human ClC\u2010Kb channel in mammalian cells impedes the functional study of <jats:italic>CLCNKB<\/jats:italic> mutations, and the underlying cause is still unclear.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>The human ClC\u2010Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C\u2010terminal in\u2010frame green fluorescent protein fusion may slow down the proteasome\u2010mediated proteolysis. Barttin co\u2010expression necessarily improves the stability, membrane trafficking and gating of ClC\u2010Kb.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p><jats:italic>CLCNKB<\/jats:italic> mutations in barttin\u2010binding sites, dimer interface or selectivity filter often have severe functional consequences.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>The remaining chloride conductance of the ClC\u2010Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS.<\/jats:p><\/jats:list-item>\n<\/jats:list><\/jats:p><\/jats:sec><jats:sec><jats:title>Abstract<\/jats:title><jats:p>Mutations in the <jats:italic>CLCNKB<\/jats:italic> gene encoding the human voltage\u2010gated chloride ClC\u2010Kb (hClC\u2010Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC\u2010Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype\u2013phenotype association has not been established. In this study, we found that hClC\u2010Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In\u2010frame fusion of green fluorescent protein (GFP) to the C\u2010terminus of the channel may ameliorate proteasome degradation. Co\u2010expression of barttin increased protein abundance and membrane trafficking of hClC\u2010Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC\u2010Kb\u2010GFP. Most <jats:italic>CLCNKB<\/jats:italic> mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well\u2010described functional consequences and clinical presentations for genotype\u2013phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC\u2010Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C\u2010terminus of hClC\u2010Kb improves protein expression. The functional severity of the <jats:italic>CLCNKB<\/jats:italic> mutation is an important determinant of the phenotype in classic BS.<\/jats:p><\/jats:sec>","DOI":"10.1113\/jp274344","type":"journal-article","created":{"date-parts":[[2017,5,27]],"date-time":"2017-05-27T09:22:41Z","timestamp":1495876961000},"page":"5573-5586","source":"Crossref","is-referenced-by-count":30,"title":["Functional severity of <i>CLCNKB<\/i> mutations correlates with phenotypes in patients with classic Bartter's syndrome"],"prefix":"10.1113","volume":"595","author":[{"given":"Chih\u2010Jen","family":"Cheng","sequence":"first","affiliation":[{"name":"Department of Medicine, Division of Nephrology, Tri\u2010Service General Hospital National Defense Medical Center  Taipei 114 Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi\u2010Fen","family":"Lo","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Nephrology, Tri\u2010Service General Hospital National 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