Wang H et al. (2013), Novel dicarboxylate selectivity in an insect gl...

XB-ART-47981

PLoS One 2013 Jan 01;88:e70947. doi: 10.1371/journal.pone.0070947.

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Novel dicarboxylate selectivity in an insect glutamate transporter homolog.

Wang H , Rascoe AM , Holley DC , Gouaux E , Kavanaugh MP .

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Mammals express seven transporters from the SLC1 (solute carrier 1) gene family, including five acidic amino acid transporters (EAAT1-5) and two neutral amino acid transporters (ASCT1-2). In contrast, insects of the order Diptera possess only two SLC1 genes. In this work we show that in the mosquito Culex quinquefasciatus, a carrier of West Nile virus, one of its two SLC1 EAAT-like genes encodes a transporter that displays an unusual selectivity for dicarboxylic acids over acidic amino acids. In eukaryotes, dicarboxylic acid uptake has been previously thought to be mediated exclusively by transporters outside the SLC1 family. The dicarboxylate selectivity was found to be associated with two residues in transmembrane domain 8, near the presumed substrate binding site. These residues appear to be conserved in all eukaryotic SLC1 transporters (Asp444 and Thr448, human EAAT3 numbering) with the exception of this novel C. quinquefasciatus transporter and an ortholog from the yellow fever mosquito Aedes aegypti, in which they are changed to Asn and Ala. In the prokaryotic EAAT-like SLC1 transporter DctA, a dicarboxylate transporter which was lost in the lineage leading to eukaryotes, the corresponding TMD8 residues are Ser and Ala. Functional analysis of engineered mutant mosquito and human transporters expressed in Xenopus laevis oocytes provide support for a model defining interactions of charged and polar transporter residues in TMD8 with α-amino acids and ions. Together with the phylogenetic evidence, the functional data suggest that a novel route of dicarboxylic acid uptake evolved in these mosquitos by mutations in an ancestral glutamate transporter gene.

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Species referenced: Xenopus laevis
Genes referenced: dct slc1a1

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	Figure 2. Transport activity of wild type CuqDCT.(A) Current recordings from a representative voltage-clamped oocyte (−60 mV) expressing wild-type CuqDCT showing concentration-dependence of responses induced by superfusion of L-glutamate (top) or succinate (bottom) at concentrations indicated above trace. Lack of response to 300 µM succinate in representative uninjected oocyte is also shown. (B) Summary of kinetic data for oocytes expressing for wild type CuqDCT showing fits of data to the Michaelis-Menten equation reflecting Km values for dicarboxylic acids approximately two orders of magnitude lower than for L-Glu. No significant difference were observed in Imax values. (C) CuqDCT-mediated currents clamped at different voltages were induced by the 100 uM L-glutamate, L-aspartate, succinate, malate and α-ketoglutarate. (D) Radiolabel [3H] succinate and L-glutamate (10 uM) uptake assay for oocytes expressing wild type CuqDCT. Uninjected oocytes were used as control. Error bars represent SEM, n≥3.
	Figure 3. The mutation N428D in CuqDCT either alone or in the C440S background changed substrate selectivity.The apparent affinities of the mutants for L-glutamate (A) were increased approximately two orders of magnitude (Km shift from 1.1±0.1 mM to 13.8±1.3 µM), while the apparent affinity for malate (B) was decreased (Km shift from 11±1 µM to 8.8±1.5 mM). (C) Comparison of the uptake of 10 µM [3H] L-glutamate and [3H] succinate for oocytes expressing the wild type CuqDCT or double mutant N428D/C440S. (D) Inward currents are induced by addition of 20 mM extracellular K+ in uninjected oocytes and in oocytes expressing wild-type CuqDCT, while oocytes expressing comparable levels of N428D/C440S mutant (fluorescence micrographs below) respond with outward currents (n≥3 for each group).
	Figure 4. Comparison of EAAT3 wild-type and D444S/T448A mutant transporter currents.Concentration-dependence of currents in response to L-glutamate (A) or succinate (B) in oocytes expressing wild-type EAAT3 or mutant D444S/T448A. Curves show fit of data to the Michaelis-Menten equation. No outward current was induced by addition of extracellular K+ in oocytes at −20 mV expressing mutant D444S/T448A, in contrast to wild type EAAT3 (B inset). Comparison of the voltage-dependence of wild type EAAT3 and D444S/T448A mutant currents induced by either 1 mM L-glutamate (C) or 10 mM succinate (D). In C and D, current amplitudes were normalized to the response at −80 mV. Error bars represent SEM, n≥3.
	Figure 5. Substrate binding domain models.Binding domain models for human EAAT3 (A,B) and CuqDCT (C,D) (see methods). The models highlight key residues in TMD8 (yellow sticks) and substrate (orange sticks). Conserved side chains in both transporters (R447/R431 and N451/N435) proposed to coordinate distal and proximal carboxylate groups of substrates by electrostatic interaction and H-bonding, respectively. Differences in the key residues (T448/A432 and D444/N428) may contribute to selectivity changes by altering interactions with substrate. Schematic diagrams generated by LIGPLOT indicate possible hydrogen bond interactions between L-glu and EAAT3 (B), and malate and CuqDCT (D).
	Figure 1. Sequence analysis of eukaryotic and prokaryotic SLC1 transporters.(A) Sequence alignment of TMD8 region from human, insect, bacterial, and archael transporters. CuqDCT, CuqEAAT, AeaDCT, and AeaEAAT represent the EAAT and DCT orthologs from Culex quinquefasciatus and Aedes aegypti, respectively. AngEAAT1 and AngEAAT2 represent the transporters from Anopheles gambiae. Drosophila EAAT1/2 are from Drosophila melanogaster, Gltph from Pyrococcus horikoshii, and DctA is from Bacillus subtilis. Highlighted TMD8 residues involved in substrate binding are labeled above using human EAAT3 numbering. R447 and N451 are conserved in all EAAT-like orthologs, while D444 and T448 are changed to asparagine and alanine, respectively in the CuqDCT and AeaDCT mosquito transporters. Sequence alignments were performed with ClustalX2 and Jalview. (B) The phylogenetic relationship of the EAAT and DCT proteins from Culex quinquefasciatus and Aedes aegypti mosquito species together with the two EAATs from Drosophila (ClustalX2 based alignment).

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