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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.

SUBMITTER: Oshiro N

PROVIDER: S-EPMC1622917 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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

Oshiro Naomi N Pajor Ana M AM

Biochimica et biophysica acta 20060516 6

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 domain ...[more]

PMID: 16787639

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Project description:Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637-651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (DeltaF), which suggested that this site lies in an environment that is affected by conformational change in the protein. DeltaF was substrate dependent (no DeltaF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1-18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.

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Dataset Information

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

Publications

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

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