Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1
| dc.contributor.advisor | Ana M. Pajor | 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 |
| 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.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.identifier.other | etd-04102003-012738 | en_US |
| dc.identifier.uri | http://hdl.handle.net/2152.3/95 | |
| 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.genre | dissertation | en_US |
| dc.type.material | text | en_US |
| thesis.degree.department | Biochemistry and Molecular Biology | en_US |
| thesis.degree.grantor | The University of Texas Medical Branch | en_US |
| thesis.degree.level | Doctoral | en_US |
| thesis.degree.name | PhD | en_US |