Transient Kinetics Reveal Mechanism and Voltage Dependence of Inhibitor and Substrate Binding to Glutamate Transporters

Abstract

Plasma−membrane glutamate transporters of the excitatory amino acid transporter (EAAT) family are important for maintaining a low glutamate concentration in the extracellular space of the mammalian brain. Glutamate is believed to be transported in its negatively charged form and energetically driven by the cotransport of three sodium ions, at least two of which are bound within the dielectric of the membrane. It was hypothesized that binding of substrates and competitive inhibitors is also electrogenic because the binding site is located near the center of the membrane. To test this hypothesis, we rapidly applied a low-affinity competitive inhibitor, kainate, to the glutamate transporter subtype EAAT2, resulting in outward transient current caused by movement of net negative charge of the inhibitor into the low dielectric of the protein/membrane. Consistent with these data, rate constants for inhibitor dissociation and binding were also voltage dependent. Our results are supported by electrostatic calculations and molecular dynamics simulations of spontaneous substrate dissociation, showing that the substrate and inhibitor binding site is located within the membrane environment of low dielectric constant. Charge movement caused by binding of negatively charged amino acid substrate is compensated by the charge of cotransported Na + ion(s), thus preventing inhibition of substrate binding at negative membrane potentials. This charge compensation mechanism may be relevant for other Na + -driven transporters which recognize negatively charged substrates.

A K+/Na+ co-binding state: Simultaneous versus competitive binding of K+ and Na+ to glutamate transporters

“…Currents associated with glutamate transporters were measured in the whole-cell current recording configuration. Whole-cell currents were recorded with an EPC7 patch-clamp Amplifier (ALA Scientific, Westbury, NY) under voltage-clamp conditions (4). The resistance of the recording electrode was 2-3 megaohms.…”

“…Glutamate uptake is energetically coupled to co-transport of three sodium ions and one proton in exchange for one K ϩ ion (1,3). Because glutamate is thought to be transported in the negatively charged form (3)(4)(5), this stoichiometry predicts that two positive charges move into the cell with each glutamate molecule, giving rise to a coupled transport current (5,6). Consistent with this prediction, transport current induced by glutamate application to glutamate transporter (excitatory amino acid transporter (EAAT))-expressing cells has been observed in many reports (7)(8)(9)(10).…”

Interaction of the neutral amino acid transporter ASCT2 with basic amino acids

“…We speculate that the amino acid binding site is located partially within the transmembrane electric field, leading to attraction of a positively charged substrate to the binding site by an inside-negative membrane potential. Similar conclusions were reached for the binding of negatively charged amino acids and inhibitors to the acidic amino acid transporters (EAATs) sharing the same solute carrier family with the ASCTs [54]. Thus, it is not unexpected that the binding mechanisms of substrates to the extracellular facing binding site are conserved within the family members.…”

Observing spontaneous, accelerated substrate binding in molecular dynamics simulations of glutamate transporters

“…In [27], substrate dissociation was studied at varying membrane potentials. The more negative the membrane potential, the faster the substrate was released to the aqueous solution.…”

“…In addition to the glutamate translocation process [14,24], the structural mechanism of substrate interaction with the binding site, including time-resolved mechanisms of binding and dissociation, have been investigated in molecular dynamics (MD) simulation studies [16,[25][26][27][28]. The successive steps of gate opening and closing, substrate recognition and eventual binding occur in nanoseconds [18].…”