Conformational changes in CIC chloride transporters

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

This project continued studying conformational changes in ClC-type chloride transport proteins. The ClC family of chloride-conducting ion channels and transporters is involved in a host of biological processes; these channels maintain the resting membrane potential in skeletal muscle, modulate excitability in central neurons, and are involved in the homeostasis of pH in a variety of intracellular compartments. Despite their physiological importance, the mechanisms by which these proteins function are poorly understood. We are attempting to understand the functional properties of these proteins by examining several family members, including both eukaryotic and prokaryotic homologs. Our current focus in this area is to observe the flux due to a single ClC transporter in a single proteoliposome. We are doing this using single molecule methods to probe the transport activity of individual ClC transport proteins. We have now reproducibly observed ClC-mediated fluxes in individual liposomes and have observed proton transport through a ClC chlroide-proton antiporter driven by opposing movement of Cl-. Using mutant ClCs with altered transport processes, we are using this assay to probe fundamental aspects of the transport mechanism. In addition, we have been exploring a patient mutation in another CLC, ClC-7, which is important in lysosomal acidification. This mutation leads to a disease different from those caused by other ClC-7C defects. We have been investigating the functional effects of the mutation by expressing WT and mutant proteins (with additional mutations that retarget the protein to the cell surface) in Xenopus oocytes or cultured mammalian cells and analyzing the properties of the resulting currents. These results give insight into the mechanism of the defect in patients and its underlying protein cause. Finally, in a somewhat unrelated project, we have participated in a collaboration to explore the relationships between structure and function in a mammalian dicarboxylate transporter, a member of a family long of interest to the lab. In collaboration with Da-Neng Wang's lab at NYU we determined the structure of this protein and demonstrated that a pharmacologic inhibitor works by pinning the transport domain in one place so it cannot move in the ways required by transport.