Transmembrane helices 3 and 4 are involved in substrate recognition by the Na+/dicarboxylate cotransporter, NaDC1.
The Na(+)/dicarboxylate cotransporters (NaDC1) from mouse (m) and rabbit (rb) differ in their ability to handle glutarate. Substrate-dependent inward currents, measured using two-electrode voltage clamp, were similar for glutarate and succinate in Xenopus oocytes expressing mNaDC1. In contrast, currents evoked by glutarate in rbNaDC1 were only about 5% of the succinate-dependent currents. To identify domains involved in glutarate transport, we constructed a series of chimeric transporters between mouse and rabbit NaDC1. Although residues found in multiple transmembrane helices (TM) participate in glutarate transport, the most important contribution is made by TM 3 and 4 and the associated loops. The R(M3-4) chimera, consisting of rbNaDC1 with substitution of TM 3-4 from mNaDC1, had a decreased K(0.5)(glutarate) of 4 mM compared with 15 mM in wild-type rbNaDC1 without any effect on K(0.5)(succinate). The chimeras were also characterized using dual-label competitive uptakes with (14)C-glutarate and (3)H-succinate to calculate the transport specificity ratio (TSR), a measure of relative catalytic efficiency with the two substrates. The TSR analysis provides evidence for functional coupling in the transition state between TM 3 and 4. We conclude that TM 3 and 4 contain amino acid residues that are important determinants of substrate specificity and catalytic efficiency in NaDC1.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-0645, USA.
Naomi
N
Oshiro
Steven C
SC
King
Ana M
AM
Pajor
D000818:Animals; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005977:Glutarates / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D017433:Protein Structure, Secondary; D011817:Rabbits; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
D029382:Dicarboxylic Acid Transporters; D005977:Glutarates; D050506:Mutant Chimeric Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; C490479:Slc13a2 protein, mouse; D027981:Symporters; C035736:glutaric acid