Detecting Ligand-Induced Motions in Pentameric Ligand-Gated Ion Channels

Abstract

Chemical signaling in the brain and periphery relies on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). While recent high resolution crystal structures of pLGICs in apparently closed, open and desensitized channel conformations are providing invaluable insights into possible activation mechanisms, whether these static protein structures, solved in detergent micelles, accurately capture the conformational gating transitions that a functional pLGIC undergoes when embedded in a lipid bilayer is unknown. Using site-directed spin labeling of a prototypical pentameric ligand-gated ion channel, GLIC, and double electron-electron resonance (DEER) spectroscopy, we are mapping motions underlying ligand-driven gating transitions in functional channels reconstituted into liposomes. In regions at the boundary between the ligand binding domain (loops 2 and 9) and the channel domain (M2-M3 loop), we found that proton-driven gating transitions induced a large movement of loop 9 and an immobilization of loop 2. In the extracellular β-sandwich domain, spin probes attached to the inner beta-sheet moved closer together at pH 3.0, suggesting that the extracellular binding domain of GLIC tilts radially inward when the channel transitions from a closed to open/desensitized states. These data, combined with recently solved structures, expand our understanding of signaling mechanisms underlying pLGIC gating transitions.