Ala-504 is a determinant of substrate binding affinity in the mouse Na+/dicarboxylate cotransporter

Abstract

The Na + /dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K 0.5 for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K 0.5 for both adipate and succinate.

Threonine-509 Is a Determinant of Apparent Affinity for Both Substrate and Cations in the Human Na+/Dicarboxylate Cotransporter

“…The amino acid at position 509/512 is also involved in binding other substrates. In a study of mouse NaDC1, we found that serine replacement of the equivalent position at Ala-504, produced a more rabbit-like phenotype with decreased affinity for succinate and adipate, although the reverse mutation in rabbit (rbS512A) was not sufficient to increase affinity for adipate (18). The S512C mutant of rbNaDC1 was not expressed on the cell surface, possibly because of protein misfolding (10).…”

“…The hydroxyl group of serine or threonine does not appear to be required for function since the mouse NaDC1 contains alanine at this position, and the S512A mutant of rbNaDC1 does not exhibit any change in functional properties (18). Possibly the size or volume of the side chain at this position affects the substrate binding site in NaDC1.…”

Differential interaction of dicarboxylates with human sodium-dicarboxylate cotransporter 3 and organic anion transporters 1 and 3

“…These include, in rabbit NaDC1, Lys-84 (22), four amino acids around Ser-260 (transmembrane helix H5), Arg-349 (H7), Asp-373 (H8), Glu-475 (H9) (summarized in Ref. 20), and Ser-512 (H10) (19). Thus, it is mainly the COOH-terminal part of NaDC3 that is involved in binding and transporting dicarboxylates.…”

Functional Reconstitution of SdcS, a Na + -Coupled Dicarboxylate Carrier Protein from Staphylococcus aureus

“…Kinetic constants for SdcS under the above-mentioned conditions were determined for each of three independent trials and are reported in the text as means Ϯ standard errors. Such substrate preference differences within the DASS family are well documented (5,8,24,25,31) and imply that the binding pockets of these transporters have diverged sufficiently to accommodate compounds that deviate slightly from the substrate template structure.…”