Substrate and Sodium Binding Energetics to a “Straight-Jacketed” Glutamate Transporter Homologue

Abstract

Glutamate transporters are secondary active transporters that couple the trans-membrane movement of the substrate to the movement ions, mainly sodium. To achieve this, the transporters alternate between outward- and inward-facing conformations, in which the substrate and ion binding sites are accessible to the extracellular and cytoplasmic sides of the membrane, respectively. Importantly, the substrate binding properties of these states and the extent to which they are modulated by sodium ions remain largely unknown. To shed light on these questions, we used a thermophilic archaeal homologue of the glutamate transporters, GltPh, solubilized in detergent and purified to homogeneity. In order to characterize the binding energetics of the isolated outward- and inward-facing states, the transporters were conformationally constrained in these states by cysteine cross-links and probed by isothermal titration calorimetry and fluorescence spectroscopy. Strikingly, both states demonstrate similar affinity for the substrate at 25 °C, which is steeply dependent on sodium concentration and increases by five orders of magnitude in the presence of 1 to 60 mM sodium. Sodium binding in the absence of substrate can be detected spectroscopically and is characterized by low affinity in both states. Interestingly, the enthalpy and the heat capacity changes upon substrate binding are different in the two states, suggesting that distinct conformational changes underlie the reactions. Using the experimentally determined temperature dependences of the thermodynamic parameters, we extrapolate the substrate binding affinities to the physiological temperatures near 100 °C for GltPh. We find that under these conditions the inward-facing state binds substrate significantly weaker than the outward-facing state.