Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1.
Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopus oocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.
Pajor
A M
AM
Department of Physiology, University of Arizona, College of Medicine, Tucson, AZ 85724, USA.
Sun
N
N
Valmonte
H G
HG
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D029382:Dicarboxylic Acid Transporters; D006639:Histidine / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D029363:Organic Anion Transporters, Sodium-Dependent; D017354:Point Mutation; D011817:Rabbits; D013329:Structure-Activity Relationship; D027981:Symporters; D014981:Xenopus
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D008565:Membrane Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; C092524:SLC13A2 protein, human; D027981:Symporters; D006639:Histidine