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 in Switzerland, we are forming 2d crystals of this protein under a series of conditions to reveal the structural changes underlying this conformational change. We are currently studying the transport mechanism in two eukaryotic ClC antiporters, ClC-4 and ClC-7. The endosomal ClC, ClC-4, 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. We are now following up on these studies to understand the detailed interaction between divalent metal ions and the ClC-4 transporter and to use this interaction as a tool to probe the transport mechanism. 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 tranport properties using electrophysiology.
|Mindell, Joseph A (2010) Structural biology. The Tao of chloride transporter structure. Science 330:601-2|
|Osteen, Jeremiah D; Mindell, Joseph A (2008) Insights into the ClC-4 transport mechanism from studies of Zn2+ inhibition. Biophys J 95:4668-75|