title: Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis.
abstract: Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.
authors: Division of Nephrology, Department of Medicine, University of California, Los Angeles, CA 90095-1689, USA. quzhu@mednet.ucla.edu
authors: Quansheng
authors: Q
authors: Zhu
authors: Xuesi M
authors: XM
authors: Shao
authors: Liyo
authors: L
authors: Kao
authors: Rustam
authors: R
authors: Azimov
authors: Alan M
authors: AM
authors: Weinstein
authors: Debra
authors: D
authors: Newman
authors: Weixin
authors: W
authors: Liu
authors: Ira
authors: I
authors: Kurtz
mesh_terms: D000141:Acidosis, Renal Tubular / Q000235:genetics / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D002254:Carbonates / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009566:Nitrates / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010641:Phenotype; D012964:Sodium / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D001639:Bicarbonates; D002254:Carbonates; D008698:Mesylates; D009566:Nitrates; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C093171:methanethiosulfonate ethylammonium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; D012964:Sodium; D005033:Ethylmaleimide
keywords: CO32-; NBCe1-A; T485S; electrogenicity; pRTA
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