Voltage Dependent Inhibitor Binding to Plasma-Membrane 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, energetically driven by the co-transport of three sodium ions. At least two of these sodium ions are bound within the dielectric of the membrane, resulting in voltage dependent association and dissociation kinetics. It was speculated that glutamate binding is also electrogenic because the binding site of the transported substrate, glutamate, is located somewhat close to the center of the membrane. Furthermore, it was hypothesized that competitive inhibitor binding is voltage dependent for the same reason. Here, 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. Consistently, inhibitor dissociation kinetics are voltage dependent, accelerating with increasingly negative transmembrane potential. In contrast, binding kinetics of a high-affinity, slow binding inhibitor, TFB-TBOA, showed the opposite voltage dependence compared to dissociation. Consistent with previous studies, our results show that the substrate and inhibitor binding site is located within the membrane environment with low dielectric constant. This work was supported by the National Science Foundation Grant 1515028 awarded to C.G.