Evolution of Ion Specificity in Glutamate Transporters

Abstract

A family of secondary active glutamate transporters imports glutamate into the cell through coupling to electrochemical gradients. In mammals, these proteins clear the synaptic cleft of excess neurotransmitter, while in prokaryotes they serve in nutrient uptake. In prokaryotes, aspartate/glutamate transporters appear to couple transport to either exclusively protons or sodium. While the mechanism of sodium-coupling is well-established for an archaeal aspartate transporter homolog (GltPh), the basis of proton-coupling is unknown. Based on evolutionary and structural analysis, we have designed two point mutations in GltPh that introduce increased transport rates in the presence of a proton gradient. The mutant protein still requires the presence of sodium, indicating that the mutant protein may be an intermediate between sodium and proton-coupled transporters. Upon aspartate binding, the mutant protein does not appear to bind or release protons, suggesting that pH-dependent transport affects the transition rate between inward and outward-facing states. Interestingly, at high sodium concentrations, the mutant protein binds aspartate in a more complex binding mode than wild-type GltPh, potentially due to a change in distribution of structural states. Further investigation into the dynamics of the proton-coupled GltPh mutant may reveal the general mechanism of proton-coupled transport in prokaryotic aspartate/glutamate transporters.