This research aims to characterize the function of the CIC transporter EriC, the only CIC member currently amenable to both structural and functional studies, in order to better understand the molecular mechanisms underlying chloride and proton transport. This will ultimately help us to understand the similarities and differences between CIC members.
In Specific Aim 1, I will map the permeation pathway of EriC by mutating candidate titratable residues in the structure and testing whether proton coupling is disrupted. I hypothesize that the permeation pathway is either through the dimer interface or near the chloride permeation pathway, both of which are testable hypotheses.
In Specific Aim 2 a, I will characterize the inhibition of EriC by the nonspecific inhibitor DIOS and localize the DIDS reactive site to allow DIDS to be used as a tool to study the function of EriC.
In Specific Aim 2 b, I will develop a high-throughput fluorescence assay to screen for potent inhibitors of EriC, which will be critical for thorough characterization of this transporter and are currently lacking for CIC members in general.
In Specific Aim 3, I will determine the rates of efflux and exchange in oriented vesicles in order to determine the rate limiting step(s) in coupled transport.
