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.
Highlights
Radioactive Transport and Transport Currents by Met-367 Mutants—The conserved methionine residue at position 367 of EAAC1 was mutated to residues with smaller or larger side chains, such as cysteine and serine or phenylalanine, respectively, and to leucine, whose side chain is roughly the same size as methionine
Mutation of Met-367 of the neuronal glutamate transporter EAAC1, a conserved residue located in the unwound portion of TM7 [14], leads to changes in the substrate selectivity (Figs. 1– 4 and Table 1)
These observations, together with the observation that substrates and the nontransportable substrate analogue TBOA protects against sulfhydryl modification of the M367C mutant (Fig. 8), suggest that Met-367 is located near the substrate in the binding pocket of EAAC1
Summary
We ask the question of whether in the eukaryotic glutamate transporters this methionine interacts with bound blocker and substrate by probing whether the mutation of Met-367 of EAAC1 results in changes of selectivity and apparent affinity of blocker and substrates
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