Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1.
The role of cationic amino acids in the Na(+)/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased K(m) for succinate of 1.8 mM, compared with 0.3 mM in the wild-type NaDC-1. The R349A mutant had Na(+) and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.
Pajor
A M
AM
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641, USA. ampajor@utmb.edu
Kahn
E S
ES
Gangula
R
R
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002412:Cations; D018076:DNA, Complementary; D029382:Dicarboxylic Acid Transporters; D005260:Female; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; D014982:Xenopus laevis
D000596:Amino Acids; D002352:Carrier Proteins; D002412:Cations; D018076:DNA, Complementary; D029382:Dicarboxylic Acid Transporters; D008565:Membrane Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; C092524:SLC13A2 protein, human; D027981:Symporters