Arginine Movement in Synergistic Coupling of Substrate and Ion Binding to Glutamate Transporter Homologue

Abstract

Glutamate transporters are membrane pumps, which catalyze reuptake of the neurotransmitter from the brain synapses driven primarily by symport of sodium ions. A bacterial homologue, GltPh is a sodium/aspartate symporter, for which the crystal structures of sodium and substrate bound states have been determined. The thermodynamic studies have demonstrated that binding of sodium ions and aspartate to the transporter are coupled, such that the affinity for the substrate is much higher in the presence of the ions and vice versa. However, the ions are not directly coordinated by the substrate and the molecular mechanism of coupling remains largely unknown. Arginine 397 interacts with the side chain carboxylate of the substrate in the transporter crystal structures, is highly conserved among glutamate transporters, and has been shown in functional studies to be a key residue for substrate binding in mammalian homologues. To probe how the formation of the substrate-binding site affects sodium binding, we generated R397A mutant. We show that, as expected, R397A mutant has a low substrate affinity even in the presence of 100 mM sodium, measured by the isothermal titration calorimetry. Remarkably, in the absence of aspartate the sodium binding affinity for R397A is increased by ∼10 fold, compared to the wild type transporter, as measured by a fluorescence assay in vitro. Furthermore, substrate does not significantly increase the affinity of the mutant transporter for the ions. We suggest that arginine 397 in the wild type GltPh interferes with the sodium binding site or sites in the absence of the substrate and that its re-orientation upon substrate coordination relieves this interference. We hypothesize that these events are key to the mechanism of the allosteric coupling between substrate and ion binding.