Detecting Conformational Changes In The Bacterial Glutamate Transporter Homolog GltPh Using EPR Spectroscopy

Abstract

Abnormal glutamate transporter function is implicated in Parkinson's disease, Alzheimer's disease, cerebral ischemia, epilepsy, and amyotrophic lateral sclerosis, underscoring the importance of understanding how these transporters function. Our research is centered on elucidating the structural and functional properties of glutamate transporters to reveal novel approaches for treating these various neuropathological conditions. A major advance in this field was the elucidation of the crystal structure of a bacterial glutamate transporter homolog, Pyrococcus horikoshii (GltPh) (Yernool et al., 2004). More recently, evidence for conformational changes in the putative extracellular gate (hairpin loop 2: HP2) was provided by crystallizing GltPh in the presence of the non-transportable blocker, TBOA (Boudker et al., 2007), rendering HP2 unable to properly close due to steric restrictions. In addition, in both the glutamate-bound and TBOA-bound crystal structures, excess non-protein electron density was found occluded in a pocket between hairpin 1 (HP1; putative internal gate), transmembrane domain 7a (TM7A), and transmembrane domain 8 (TM8). This was interpreted as being trapped solvent, and suggested that the trapped solvent was the result of the fact that the putative internal gate (HP1) was closed in both structures. Further conformational change was speculated to expand this solvent-filled cavity, providing a pathway for glutamate to reach the cytoplasm, potentially along the polar face of TM8. Therefore, using site-directed spin-labeling electron paramagnetic spectroscopy (SDSL-EPR) on GltPh, we are working to define the conformational changes that occur in both the extra- and intracellular gates during the glutamate transport process, and to define the pore-like region that allows glutamate access to the cytosol.