Conformational changes in CIC chloride transporters

Mindell, Joseph

Abstract

This project continued studying conformational changes in ClC-type chloride channel proteins. The ClC family of chloride-conducting ion channels 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 channels 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 in collaboration with Dimitrios Stamou at the University of Copenhagen. Toward this end we have developed a robust bulk assay of chloride-coupled proton transport, and then subjected individual liposomes to similar conditions. We have now reproducibly observed ClC-mediated fluxes in individual liposomes and have obeserved complex pH variation, suggesting that current models do not fully explain the function of the CLC protein. IN addition, over the past year 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 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 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.