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 combination of methods to analyze the functional mechanisms of these proteins. Previously, we used fluorescence methods to demonstrate the existence a transport-related conformational change in a bacterial ClC antiporter. Currently, in collaboration with Henning Stahlberg at UC Davis, 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 we began to study the transport mechanism in a eukaryotic ClC antiporter, ClC-4. This endosomal ClC is sent to the plasma membrane when heterologously expressed in Xenopus oocytes allowing analysis using electrophyiological methods. We searched for molecular tools that might be useful in probing the transport process and found that Zn2+ and Cd2+ both inhibit ClC-4 currents. We used site-directed mutagenesis to locate the Zn2+ binding site and found intruiging interactions between Zn2+ binding and the ion transport process. These finding reveal movements in a part of the protein that has not been previously implicated in transport-related conformational changes.