Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1

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Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1

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dc.contributor.advisor Ana M. Pajor en_US
dc.creator Hongyan Li en_US
dc.date.accessioned 2011-12-20T16:04:36Z
dc.date.available 2003-05-21 en_US
dc.date.available 2011-12-20T16:04:36Z
dc.date.created 2003-04-10 en_US
dc.date.issued 2003-01-28 en_US
dc.identifier.other etd-04102003-012738 en_US
dc.identifier.uri http://hdl.handle.net/2152.3/95
dc.description.abstract In this dissertation, two cation-coupled transporters were characterized at the molecular level. The CitM transporter from Bacillus subtilis was functionally expressed and characterized in E.coli cells. The human NaSi-1 transporter (hNaSi-1) and mutants were functionally expressed in Xenopus oocytes. Antibodies against hNaSi-1 were used to investigate tissue distribution and N-glycosylation. The roles of two conserved serine residues in the transport function of hNaSi-1 were investigated using site-directed mutagenesis and radiotracer assay. \r\n\r\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM in the presence of saturating Mg2+ concentrations. CitM has a high substrate specificity for citrate. Other tested di- and tricarboxylic acids did not significantly inhibit citrate uptakes in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The transport was inhibited in more alkaline but not in acidic transport buffer and also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin, suggesting a proton-coupled transport. Valinomycin did not affect the uptake by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \r\n\r\nThe low affinity Na+/sulfate cotransporter, hNaSi-1, belongs to a specific gene family of Na+-coupled transporters that includes the high affinity hSUT-1 and the Na+-coupled dicarboxylate (NaDC) transporters. Antibodies directed against a peptide of hNaSi-1 recognized the native protein in renal membranes as well as the recombinant protein expressed in Xenopus oocytes. There is a single N-glycosylation site, Asn-591, located at the extracellular C-terminus in hNaSi-1. Site-directed mutagenesis studies of Ser-260, Ser-288 and the surrounding amino acid residues of hNaSi-1 suggested that these residues are functionally required for hNaSi-1. MTSET inhibition on sulfate uptakes by the four mutants surrounding Ser-260, T257C, T259C, T261C and L263C, was dependent on the cation and substrate used. Since the presence of sodium and sulfate triggers conformational changes during the transport cycle of hNaSi-1, the cation and substrate dependence of MTSET inhibition suggest that these four substituted cysteines move during the transport cycle. Since the four mutated residues are located in TMD-5, this transmembrane domain is also likely to participate in the conformational movement during the transport cycle of hNaSi-1. \r\n en_US
dc.format.medium electronic en_US
dc.language.iso eng en_US
dc.rights Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works. en_US
dc.subject Western blots en_US
dc.subject radiotracer uptake assay en_US
dc.subject PCR en_US
dc.subject mutagenesis en_US
dc.subject immunofluorescence en_US
dc.subject brush border membrane vesicles en_US
dc.subject biotinylation en_US
dc.subject en_US
dc.title Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1 en_US
dc.type.material text en_US
dc.type.genre dissertation en_US
thesis.degree.name PhD en_US
thesis.degree.level Doctoral en_US
thesis.degree.grantor The University of Texas Medical Branch en_US
thesis.degree.department Physiology and Biophysics en_US
dc.contributor.committeeMember Steven C. King en_US
dc.contributor.committeeMember Steven A. Weinman en_US
dc.contributor.committeeMember Luis Reuss en_US
dc.contributor.committeeMember Joel P. Gallagher en_US

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