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

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

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Grantor

The University of Texas Medical Branch

Year dc:date.issued

2003

Author dc:creator

• Hongyan Li

Advisor dc:contributor.advisor

• Ana M. Pajor

Committee members dc:contributor.committeemember

• Steven C. King
• Steven A. Weinman
• Luis Reuss
• Joel P. Gallagher

• Western blots
• radiotracer uptake assay
• PCR
• mutagenesis
• immunofluorescence
• brush border membrane vesicles
• biotinylation

Statement 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.

Language dc:language.iso

eng

Dc Identifier Other

etd-04102003-012738

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/95