Abstract
Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2-M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating.