Threonine-509 is a determinant of apparent affinity for both substrate and cations in the human Na+/dicarboxylate cotransporter.
The Na+/dicarboxylate cotransporter (NaDC1) is involved in the absorption of citric acid cycle intermediates from the lumen of the renal proximal tubule and small intestine. The NaDC1 orthologues from human (h) and rabbit (rb) exhibit differences in citrate and cation transport properties. The citrate Km and sodium KNa values are much larger in human than rabbit NaDC1. Our previous study showed that transmembrane helices (TM) 7, 10, and 11 and associated loop regions contain the amino acids that are important in determining the differences in apparent citrate affinity, whereas TM10 and 11 determine differences in apparent sodium affinity. Chimera R10 (hNaDC1 with a substitution of TM10 and associated loop from rbNaDC1) contains only four amino acid differences between rb and hNaDC1. This chimera has similar apparent affinity for succinate and sodium as the wild-type rbNaDC1, and an intermediate Km for citrate. To identify individual residues in the TM10 region that determine functional differences between rb and hNaDC1, four mutants were made in which the rabbit sequence was substituted for that of the hNaDC1. Mutants with a serine or threonine at position 509 (or 512 in rbNaDC1) in TM10 have partial changes in Km for citrate and succinate but larger changes in apparent affinity for cations and substrate specificity for four-carbon dicarboxylates. The results show that the serine or threonine at position 509 (h) or 512 (rb) is the most important determinant of functional differences in apparent affinity for substrate and cations. Furthermore, the results suggest that the cation and substrate binding sites are located in close proximity to one another in NaDC1.
Weerachayaphorn
Jittima
J
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-0645, USA.
Pajor
Ana M
AM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019343:Citric Acid / Q000737:chemistry* / Q000378:metabolism; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D019802:Succinic Acid / Q000737:chemistry* / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000737:chemistry*
D029382:Dicarboxylic Acid Transporters; D029363:Organic Anion Transporters, Sodium-Dependent; D011994:Recombinant Proteins; C092524:SLC13A2 protein, human; D027981:Symporters; D019343:Citric Acid; D013912:Threonine; D012964:Sodium; D019802:Succinic Acid