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.

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