Transport Cycle and Inhibition of Human Na+-dicarboxylate Cotransporter NaDC3

PROJECT SUMMARY The human high-affinity Na+-driven dicarboxylate transporter NaDC3 plays critical roles in physiology. The gene of the transporter, SLC13A3, is expressed in the brain as well as other tissues. Located in the plasma membrane, the transporter imports various dicarboxylates into the cell, including succinate, a-ketoglutarate (aKG) and N- acetyl-aspartate (NAA). In addition to being intermediates of the TCA cycle, dicarboxylates imported by NaDC3 also function as signaling molecules in the cell. In the brain, NAA functions as a precursor for the synthesis of both neurohormones and lipids and as an osmolyte in cell volume regulation and in axon-glial signaling. The transporter is involved in a number of disorders, including the lethal Canavan disease, an early-onset CNS condition with “spongiform” vacuolation of white matter in the brain, caused by the overloading of NAA in glial cells. Inhibition of the dicarboxylate transporter is a possible treatment, and small-molecule inhibitors have been identified. In this project, we aim (1) to characterize the structural basis of NaDC3’s substrate specificity. (2) to study inhibition mechanisms. (3) to investigate the conformational changes between the outward- and inward- facing transitions. (4) to understand the sodium—substrate energy coupling. By accomplishing these aims, we will be able to understand the molecular mechanism of the human NaDC3 over its complete transport cycle, including its Co—Ci conformational transition and substrate—sodium coupling mechanism. Comparison of the inhibitor-binding sites in NaDC3 and other proteins will aid in the design of NaDC3 specific inhibitors to help patients suffering from Canavan disease.