PMID:20424168

Citation	Rosental, N and Kanner, BI (2010) A conserved methionine residue controls the substrate selectivity of a neuronal glutamate transporter. J. Biol. Chem. 285:21241-8
Abstract	Glutamate transporters located in the brain maintain low synaptic concentrations of the neurotransmitter by coupling its flux to that of sodium and other cations. In the binding pocket of the archeal homologue Glt(Ph), a conserved methionine residue has been implicated in the binding of the benzyl moiety of the nontransportable substrate analogue threo-beta-benzyloxyaspartate. To determine whether the corresponding methionine residue of the neuronal glutamate transporter EAAC1, Met-367, fulfills a similar role, M367L, M367C, and M367S mutants were expressed in HeLa cells and Xenopus laevis oocytes to monitor radioactive transport and transport currents, respectively. The apparent affinity of the Met-367 mutants for D-aspartate and L-glutamate, but not for L-aspartate, was 10-20-fold reduced as compared with wild type. Unlike wild type, the magnitude of I(max) was different for each of the three substrates. D-glutamate, which is also a transportable substrate of EAAC1, did not elicit any detectable response with M367C and M367S but acted as a nontransportable substrate analogue in M367L. In the mutants, substrates inhibited the anion conductance as opposed to the stimulation observed with wild type. Remarkably, the apparent affinity of the blocker D,L-threo-beta-benzyloxyaspartate in the mutants was similar to that of wild type EAAC1. Our results are consistent with the idea that the side chain of Met-367 fulfills a steric role in the positioning of the substrate in the binding pocket in a step subsequent to its initial binding.
Links	PubMed PMC2898430 Online version:10.1074/jbc.M109.087163
Keywords	Amino Acid Transport System X-AG/chemistry; Amino Acid Transport System X-AG/metabolism; Animals; Biological Transport; Cloning, Molecular; Electrophysiology/methods; Excitatory Amino Acid Transporter 3/chemistry; Genetic Vectors; HeLa Cells; Humans; Methionine/chemistry; Mutation; Oocytes/metabolism; Rabbits; Substrate Specificity; Xenopus laevis
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RABIT:EAA3	involved_in	GO:0070779: D-aspartate import across plasma membrane	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
RABIT:EAA3		GO:0070779: D-aspartate import	ECO:0000315:		P		Figure 1 shows uptake of substrates is reduced with mutation of Met-367.	complete CACAO 9243

References

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