Charge Compensation Mechanism of Glutamate Transport

Abstract

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na+ and one proton, and the outward movement of one K+. Glutamate, Na+ and H+ are moved to the cytosol in the translocation steps. Subsequent outward movement of K+ occurs in a separate step. Based on indirect evidence, it was suggested that outward movement of positively-charged K+ is overcompensated by the outward movement of negative charge of the binding site(s). However, no information is available on the K+-dependent reaction step(s). Here, we examined the electrostatics of the glutamate transport process by evaluating implicit membrane/solvent/ explicit transporter models using the Poisson-Boltzmann (PB) equation. The results indicate that structural changes of the transporter bound to only one cation lead to transfer of negative charge across the membrane. In order to compensate for the negatively-charged binding sites, at least two Na+ and one proton need to be bound to the transporter. Consistent with these predictions, transient currents were observed in response to steps of the transmembrane potential when K+ was the only cation present on both sides of the membrane. No K+-dependent charge movements were observed in a transporter with the mutation E373Q, which is known to be defective in the K+-dependent relocation step(s), but not K+ binding. Furthermore, rapid extracellular K+ application to EAAC1 under single turnover conditions (only K+ inside) results in outward transient current, in contrast to the expected inward current in the absence of charge overcompensation. We propose a charge compensation mechanism for the transport process, in which the C-terminal transport domain is overall negatively charged, with an apparent valence of −1.26. Our model can be used to predict the kinetics of the K+-dependent half-cycle of the glutamate transport process.