Detecting Transport-related Conformational Changes In The Glutamate Transporter Homologue, GltPh.

Abstract

Glutamate transporters (EAATs) are essential in clearing glutamate from the synapse, allowing precise control over excitatory synaptic function. Much has been learned in recent years about how these proteins function, however little is known about how the structural basis of this function; how do protein conformational changes lead to transport activity? A homolog of these proteins, GltPh from Pyrococcus horikoshii, is functionally similar to the mammalian proteins; co-transporting substrate and sodium ions whilst having an uncoupled chloride conductance. Importantly, however it has been crystallized and its structure determined, making it an excellent model for understanding how structure and function are related in the EAATs. In order for transport to proceed, conformational changes must occur that allow alternating access of the substrate binding site to both the internal and external solutions. Using fluorescein maleimide labeling and protease accessibility studies on single cysteine mutants of GltPh we have identified regions of the protein involved in this process. Changes in accessibility are seen in the presence of both aspartate and TBOA, a non-transported competitive inhibitor. Surprisingly, our accessibility studies implicate heretofore unexpected areas of the GltPh protein in the conformational changes associated with substrate and inhibitor binding. We are also further investigating functional aspects of GltPh transport using radioligand flux assays.