This was the fourth year for this project, using biochemical methods to study 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 the prokaryotic ClC's for biochemical studies of conformational changes upon activation of the transport process. Toward this goal, we have constructed a series of cysteine mutants in a critical region of the transport protein and can covalently attach fluorescent probes to these sites. We find that there are dramatic and reversible changes in fluorescence upon activation of transport at low pH, reflecting a significant conformational change in the protein at low pH. In the past year we have begun to explore this process further. We have generated and characterized mutant proteins lacking the 3 native cysteines to reduce the background labeling by some reagents. On order to probe the conformational change in more detail, we have used these constructs in preliminary characterizations of fluorescence energy transfer and in experiments using electron paramagnetic resonance. We have also reconstituted ClC-ec1 into planar lipid bilayer membranes to explore its electrical properties. Using these and other approaches we seek to further define the conformational changes underlying transport in this model ClC and thereby to gain insight into the physical processes responsible for gating and transport in mammalian ClC proteins.
| Mindell, Joseph A (2008) The chloride channel's appendix. Nat Struct Mol Biol 15:781-3 |
| Bell, Susan P; Curran, Patricia K; Choi, Sean et al. (2006) Site-directed fluorescence studies of a prokaryotic ClC antiporter. Biochemistry 45:6773-82 |
