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. In this project, we are using a the fluorescence methods to demonstrate the existence a transport-related conformational change in a bacterial ClC antiporter. Currently, in collaboration with Henning Stahlberg in Switzerland, we are forming 2d crystals of this protein under a series of conditions to reveal the structural changes underlying this conformational change. In the past year's project has moved forward with our discovery of the crystal form stable at both high and low pH. Preliminary projection maps from these crystals suggest differences in conformation between high and low pH forms of the proteins in the lipid bilayer membrane. We are currently focusing our mechanistic efforts on the lysosomal CLC, ClC-7. Recent reports demonstrate that another mammalian ClC, ClC-7, though usually targeted to lysosomes, can be retargeted to the plasma membrane by mutating a dileucine sorting motif. We have used this method to retarget ClC-7 and are studying its transport properties using electrophysiology. Our initial characterization, nearly ready for publication, examines the external pH dependence of the ClC-7 transporter and probes the relationship between the transport cycle in a novel form of gating observed in the CLC's.