My long-term research interest is to understand the structure and function of ion channels. These proteins catalyze the translocation of ions across cell membranes and are pivotal in controlling many fundamental physiological processes. In this application, I propose to continue our work on gating-permeation coupling mechanism of the Torpedo ClC-0 chloride (CI-) channel, which is considered as a prototype for the family of """"""""ClC-type"""""""" channels. These CIC channels are important in their own right because they are found in many tissues such as skeletal muscle, kidney and brain, and disruptions of these genes cause myotonia, kidney stone diseases, and developmental deficits in brain structures respectively. Moreover, the operations of these channels are particularly interesting because the gating and permeation processes are tightly coupled. In particular, the voltage dependence of muscle-type ClC channels appears to come from this gating-permeation coupling, a mechanism completely different from that in the traditional """"""""S4""""""""-type cation channels. A mechanistic study of this coupling mechanism, therefore, is of fundamental importance to understand the malfunction of ClC channel proteins defective in human diseases. Most recently, the 3-D structures of two bacterial ClC channels were solved by the MacKinnon Lab. We will take advantage of the structures from bacterial channels to explore the structural basis of the gating mechanism of CIC-0. In particular, we will study the """"""""fast gating"""""""" of this Torpedo channel, using heterologously expressed channels in Xenopus oocytes. We will first study an electrostatic interaction at the inner pore mouth known to be critical in controlling the fast gating of CIC-0. We will also investigate how a CI ion at the selectivity filter affects the fast gating. We will explore the functional role of a critical glutamate residue that appears to be important in interacting CI at the selectivity filter. Finally, we will study the gating motion that underlies the open-close transition of the channel. The results from this study will not only lead to a further understanding on the coupling of ion permeation to the fast gating in ClC-0 but will provide insight for understanding the gating of other CIC channels.
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