title: Acidic residues involved in cation and substrate interactions in the Na+/dicarboxylate cotransporter, NaDC-1.
abstract: The role of acidic amino acid residues in cation recognition and selectivity by the Na+/dicarboxylate cotransporter, NaDC-1, was investigated by site-directed mutagenesis and expression in Xenopus oocytes. Four of the residues tested, Asp-52, Glu-74, Glu-101, and Glu-332, were found to be unimportant for transport activity. However, substitutions of Asp-373 and Glu-475, conserved residues found in transmembrane domains M8 and M9, respectively, altered transport kinetics. Replacements of Asp-373 with Ala, Glu, Asn, and Gln resulted in changes in sodium affinity and cation selectivity in NaDC-1, indicating that the carbonyl oxygen at this position may play a role in the topological organization of the cation-binding site. In contrast, substitutions of Glu-475 led to dramatic reductions in transport activity and changes in transport kinetics. Substitution with Gln led to a transporter with increased substrate and sodium affinity, while the E475D mutant was inactive. The E475A mutant appeared to have poor sodium binding. Substrate-induced currents in the E475A mutant exhibited a strong voltage dependence, and a reversal of the current was seen at -30 mV. The results suggest that Glu-475 may play a role in cation binding and possibly also in mediating anion channel activity. Remarkably, mutations of both Asp-373 and Glu-475 affected the Km for succinate in NaDC-1, suggesting dual roles for these residues in determining the affinity for substrate and cations. We propose that at least one of the cation-binding sites and the substrate-binding site are close together in the carboxy-terminal portion of NaDC-1, and thus transmembrane domains M8 and M9 are candidate structures for the formation of the translocation pathway.
authors.affiliation: Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Griffith
authors.forename: A M
authors.initials: AM
authors.lastname: Pajor
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002414:Cations, Monovalent / Q000378:metabolism; D029382:Dicarboxylic Acid Transporters; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D029363:Organic Anion Transporters, Sodium-Dependent; D018408:Patch-Clamp Techniques; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013386:Succinates / Q000037:antagonists & inhibitors / Q000378:metabolism; D027981:Symporters; D014981:Xenopus
chemical_list: D002352:Carrier Proteins; D002414:Cations, Monovalent; D029382:Dicarboxylic Acid Transporters; D008565:Membrane Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; C092524:SLC13A2 protein, human; D013386:Succinates; D027981:Symporters; D001224:Aspartic Acid; D018698:Glutamic Acid; D008094:Lithium; D012964:Sodium; D000409:Alanine