Abstract
Glutamate transport is coupled to the co-transport of 3Na(+) and 1H(+) and the countertransport of 1 K(+). However, the mechanism of how this process occurs is not well understood. The crystal structure of an archaeal homolog of the human glutamate transporters, Glt(Ph), has provided the framework to begin to understand the mechanism of transport. The glutamate transporter EAAT2 is different from other subtypes in two respects. First, Li(+) cannot support transport by EAAT2, whereas it can support transport by the other excitatory amino acid transporters, and second, EAAT2 is sensitive to a wider range of blockers than other subtypes. We have investigated the relationship between the cation driving transport and whether the glutamate analogues, l-anti-endo-3,4-methanopyrrolidine-dicarboxylic acid (MPDC) and (2S,4R)-4-methylglutamate (4MG), are substrates or blockers of transport. We have also investigated the molecular basis for these differences. EAAT2 has a Ser residue at position 441 with hairpin loop 2, whereas the corresponding residue in EAAT1 is a Gly residue. We demonstrate that if the transporter has a Ser residue at this position, then 4MG and MPDC are poor substrates in Na(+), and Li(+) cannot support transport of any substrate. Conversely, if the transporter has a Gly residue at this position, then in Na(+) 4MG and MPDC are substrates with efficacy comparable with glutamate, but in Li(+) 4MG and MPDC are poor substrates relative to glutamate. This Ser/Gly residue is located between the bound substrate and one of the cation binding sites, which provides an explanation for the coupling of substrate and cation binding.
Highlights
excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3Naϩ and 1Hϩ and the countertransport of 1Kϩ (4), which provides sufficient energy to generate a 106-fold gradient of L-glutamate across the cell membrane
For EAAT1, Liϩ can partially substitute for Naϩ in driving the transport of L-glutamate but not transport of MPDC
The amino acid sequences of EAAT1, EAAT2, and GltPh are closely related in the substrate and ion binding regions (Fig. 1), but there is a Ser residue in EAAT2 within the loop region between HP2a and HP2b, whereas the corresponding residue in GltPh and the other EAATs is a Gly
Summary
Chemicals—All chemicals were obtained from Sigma unless otherwise stated. 4MG, threo-3-methylglutamate, and MPDC were obtained from Tocris (Bristol, UK). Oocytes were harvested from X. laevis, as previously described, with all procedures in accordance with the Australian National Health and Medical Research Council guidelines for the prevention of cruelty to animals. Current (I) as a function of substrate concentration ([S]) was fitted by least-squares analysis to Equation 1, I/Imax ϭS/͑EC50 ϩS͒. Where Imax represents the maximal current, EC50 is the concentration of substrate that generates a half-maximal current, and [S] is the concentration of substrate. Concentration-dependent inhibition of substrate-induced currents by blockers were fitted to Equation 2, I/Imax ϭ 1 Ϫ͑B/͑IC50 ϩB͒͒. Where [B] represents the concentration of the blocker, and IC50 is the concentration of blocker that generates half-maximal inhibition. It has previously been demonstrated that 4MG, threo-3-methylglutamate, TBOA, and kainate are competitive blockers of EAAT2 (12, 14, 15), so Ki values were calculated from IC50 values using the Cheng-Prusoff equation (16)
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