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

Show full item record

Title: Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1
Author: Hongyan Li
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
URI: http : / /hdl .handle .net /2152 .3 /95
Date: 2003-01-28

Citation

Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1. Doctoral dissertation, The University of Texas Medical Branch. Available electronically from http : / /hdl .handle .net /2152 .3 /95 .

Files in this item

Files Size Format View
dissertation(041003-final).pdf 2.014Mb application/pdf View/Open
hli.bak.pdf 0bytes application/pdf View/Open
hli.pdf 0bytes application/pdf View/Open

This item appears in the following Collection(s)

Show full item record

Search DSpace

Advanced Search

Browse