In the past year our work has focused on both GltPh, a model for the EAAT family of glutamate transporters and a new protein, vcINDY, a succinate transproter which has been implicated in longevity and obesity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We have continued our work using EPR spectroscopy to monitor conformational changes in GltPh. This work has identified local changes in the protein that may be important for coupling between the driving ion, Na+, and the substrate, aspartate. We are continuing work to identify the nature of this change. We recently reported that a extracellular loop of gltPH must be intact for effective transport. Last year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. This year we also found that only the translocation of the substrate-bound form of the protein is affected--the apo, substrate-free transporter is unaffected by 34 loop cleavage. We have performed important controls eliminating alternative explanations for these effects and a paper describing this work is under review. In the past year we also made substantial progress in work on a vcINDY, a transporter which is important for longevity in flies and is involved in obesity and insulin resistance in mammals. We performed the first successful functional reconstitution of vcINDY and directly demonstrated that it is a Na+ coupled succinate transporter and completed a comprehensive analysis of its functional properties, which was published this spring. Currently we are shifting to more mechanistic analysis with the goal of understanding the protein dynamics underlying tranpsort in this unusual protein family. We have performed an extensive analysis of the role of dimerization for the transport mechanism and begun experiments designed to analyze the mechaism by which substrate is translocated across the membrane tightly coupled to the movement of Na+ ions. We have also been making substantial progress in developing new methods to accurately determine substrate stoichiometry for transporters in general, a critical parameter required to understand mechanisms.
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