On the mechanism of proton transport by the neuronal excitatory amino acidcarrier 1

Uptake of glutamate from the synaptic cleft is mediated by high affinity tr ansporters and is driven by Na+, K+, and H+ concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracel lular pH change associated with 9 lutamate transport by combining current r ecordings from excitatory amino acid carrier 1 (EAAC1)-expressing HEK293 ce lls with a rapid kinetic technique with a 100-mus time resolution. Under co nditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the c is-side of the membrane, whereas the currents at saturating glutamate conce ntrations are hardly affected by the pH. Consistent with this, the kinetics of the pre-steady state currents, measured after saturating glutamate conc entration jumps, are not a function of the pH. In addition, we determined t he deuterium isotope effect on EAAC1 kinetics, which is in agreement with p roton cotransport but not OH- countertransport. The results can be quantita tively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and tr anslocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is similar to8. This value is shifte d to similar to6.5 when the substrate binding site is exposed to the cytopl asm.