The proposed outlines 2 complementary approaches to study the conformational changes in the gating of the ligand-gated ion channels (LGIC): (1) to discover structural elements involved in channel gating; and (2) to measure conformational changes during gating. An absolutely conserved feature in all subunits of the LGIC superfamily with the 4 transmembrane (TM) domain topology, is a proline residue in the middle of TM1. The conservation and location of TM1 proline with other natural amino acids causes the receptor to gate between closed and desensitized states. To further understand the mechanism of this phenomenon, synthetic proline analogs will be used to replace the proline and the effects of such replacement on channel gating should allow us to determine important chemistry for gating. Tom monitor conformational changes during gating, fluorescent probes will be introduced at specific sites in TM1 and simultaneous measurements of fluorescent changes and whole cell currents will be performed. Understanding the gating of LG1Cs should help us (1) understand the synaptic transmission mediated by these channels and (2) design specific therapeutic agents, many of which have already been shown to exert their effects through specific interactions with these receptor channels.
