Sodium-dependent extracellular accessibility of Lys-84 in the sodium/dicarboxylate cotransporter.
The Na(+)/dicarboxylate cotransporter transports Na(+) with citric acid cycle intermediates such as succinate and citrate. The present study focuses on transmembrane helix 3, which is highly conserved among the members of the SLC13 family. Fifteen amino acids in the extracellular half of transmembrane helix (amino acids 98-112) as well as Lys-84, previously shown to affect substrate affinity, were mutated individually to cysteine and expressed in the human retinal pigment epithelial cell line. Transport specificity ratio analysis shows that determinants for distinguishing succinate and citrate are found at amino acids Lys-84, Glu-101, Trp-103, His-106, and Leu-111. All of the mutants were tested for sensitivity to the membrane-impermeant cysteine-specific reagent (2-sulfonatoethyl) methanethiosulfonate (MTSES), but only K84C was sensitive to MTSES inhibition. The sensitivity of K84C to MTSES was greatest in the presence of sodium, and the inhibition could be prevented by addition of substrate or replacement of sodium, indicating that the accessibility of Lys-84 changes with conformational state. The substrate protection of MTSES inhibition of K84C appears to occur early in the transport cycle, before the large-scale conformational change associated with translocation of substrate. The results point to a new location for Lys-84 within the substrate access pore of the Na(+)/dicarboxylate cotransporter, either in a transmembrane helix or a reentrant loop facing a water-filled pore.
Weerachayaphorn
Jittima
J
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-0645, USA.
Pajor
Ana M
AM
D000818:Animals; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism; D002952:Citric Acid Cycle / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D020125:Mutation, Missense; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D011817:Rabbits; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D019802:Succinic Acid / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D008698:Mesylates; D029363:Organic Anion Transporters, Sodium-Dependent; D013439:Sulfhydryl Reagents; C109153:(2-sulfonatoethyl)methanethiosulfonate; D019343:Citric Acid; D012964:Sodium; D019802:Succinic Acid; D008239:Lysine