Arginine-349 and aspartate-373 of the Na(+)/dicarboxylate cotransporter are conformationally sensitive residues.
The conserved residues, Arg-349 and Asp-373, of the renal Na(+)/dicarboxylate cotransporter (NaDC-1) have been shown in our previous studies to affect substrate affinity and cation binding. In this study, amino acids surrounding Arg-349 and Asp-373 were individually mutated to cysteines and their sensitivity to methanethiosulfonate reagents (MTS) was tested. Only three of the 21 mutants were sensitive to MTS reagents: R349C, S372C, and D373C. The R349C mutant had reduced activity which was restored by chemical modification with MTSEA. The effect of MTSEA was only observed in the presence of sodium, indicating that Arg-349 is conformationally accessible. The succinate transport activity of the S372C mutant was stimulated by both MTSEA and MTSET. The D373C mutant was very sensitive to inhibition by MTSET (K(i) = 0.5 microM) in sodium buffer. The inhibition of D373C by MTSET was prevented by substrate, suggesting that the substrate-induced conformational change occludes the residue. We conclude that the accessibility of Arg-349 and Asp-373 is likely to change with the conformational states of the transport cycle.
Yao
Xiaozhou
X
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641, USA.
Pajor
Ana M
AM
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D001224:Aspartic Acid; D001665:Binding Sites; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism
D029382:Dicarboxylic Acid Transporters; D029363:Organic Anion Transporters, Sodium-Dependent; D011994:Recombinant Proteins; C092524:SLC13A2 protein, human; D027981:Symporters; D001224:Aspartic Acid; D001120:Arginine; D012964:Sodium; D003545:Cysteine