Detection of Substrate-Dependent Conformational Changes in HP1 of the Glutamate Transporter GltPh

Abstract

Glutamate acts as the primary excitatory neurotransmitter in the mammalian central nervous system. Clearance of this neurotransmitter from the synapse is accomplished by a family of glutamate transporters known as EAATs, which co-transport sodium and glutamate across the membrane. Recently, crystal structures of a related bacterial transporter, GltPh, have been solved. These structures have provided clues to the conformational changes associated with inhibitor binding as well as a large conformational change involved in switching the protein from an “outward facing” state to an “inward facing” state. However, crystallography has failed to provide data regarding the effects of sodium on the local structure of the substrate binding site. The details of how the substrate is released from the transporter are also absent. Finally, it remains undetermined which conformation(s) the protein adopts in physiological environments, and how the binding of sodium and substrate are coupled to conformational exchange. To probe these questions, we utilize the technique of site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to explore the local structure and dynamics of residues within a helical hairpin (HP1) which has been suggested to serve as an intracellular gate. EPR spectra of labeled mutants describe a conformational change within HP1 upon addition of sodium and/or aspartate. The spectral changes are localized to the tip of the hairpin, suggesting a relatively small conformational change. In order to fully interpret these structural changes, we are currently measuring the distance between doubly-labeled residues to ascertain the global conformation (outward- vs. inward-facing) of the protein. Preliminary data suggest that in vesicles, GltPh is able to adopt a conformation which resembles the most recently solved crystal structure, which has been interpreted as an inward facing form of the transporter.