Summary of distances.

<p>In roman type, adjacent (short) and nonadjacent (long) interspin distances and corresponding peak widths measured by DEER spectroscopy at pH 7.6 and pH 4.6 for GLIC mutants reconstituted into PE∶PG liposomes. In italics, adjacent (short) and nonadjacent (long) interresidue C_β–C_β distances for C26, K32, T157, K247, and P249 obtained from GLIC (PDB entry 3EHZ, pH 4.6 distances), and for aligned residues I23, L29, D158, R254, and P256 in ELIC (PDB entry 2VL0, pH 7.6 distances). Last column shows the calculated intrasubunit displacement δ values (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001714#s4" target="_blank">Materials and Methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001714#pbio.1001714.s004" target="_blank">Figure S4</a>).</p

Location of loop 2, loop 9, and M2–M3 loop in GLIC.

<p>(A) Crystal structure of GLIC (PDB entry 3EHZ) with residues C26, K32, T157, K247, and P249 shown in space-fill. The fifth subunit on the backside was removed for clarity. (B) Close-up view of an intersubunit interface highlighting the region between the extracellular and transmembrane domains and the sites spin-labeled (space-fill). (C) Positions of loop 2 and M2–M3 loop in GLIC (3EHZ, cyan) and ELIC (2VL0, red) in aligned structures. Relative to ELIC, GLIC loop 2 is shifted inward towards the channel pore-lining M2 helix (labeled), whereas GLIC M2–M3 loop is shifted outward (arrows). (D) Positions of loop 9 in GLIC (cyan) and ELIC (red) in aligned structures. Relative to ELIC, GLIC loop 9 is shifted inward toward the M2 helix (arrow).</p

Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

<div><p>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.</p></div

Detergent prevents proton-induced GLIC gating motions.

<p>(Right panel) Interspin distance distributions from model-free Tikhonov fits of X-band DEER data from GLIC T157R1 purified in detergent (DDM) micelles at pH 7.6 (black) and pH 4.6 (blue). (Left panel) The background-corrected dipolar evolution data at pH 7.6 and pH 4.6 (grey lines) and the Tikhonov fits (pH 7.6, black lines; pH 4.6, blue lines). The interspin DEER-derived distances are similar at both pH values, indicating that detergent-solubilized GLIC does not undergo proton-mediated gating motions.</p

Effects of lipids on proton-induced gating motions.

<p>X-band CW EPR spectra of T157R1 reconstituted into PE∶PG (top), PE∶PG∶cholesterol (middle), and PE∶PG∶cardiolipin (bottom) liposomes at pH 7.6 (black, resting state) and pH 4.6 (blue, desensitized). Proton-induced changes in T157R1 mobility in the presence of cholesterol were indistinguishable from those of T157R1 reconstituted in PE∶PG, whereas cardiolipin hindered gating-induced changes in T157R1 mobility.</p

Proton-induced distance changes revealed by DEER spectroscopy.

<p>(Top panel) Top-down view of GLIC crystal structure shown in ribbon representation with T157 in spacefill, black lines depict distances between adjacent and nonadjacent residues. (Left panels) Background subtracted Q-band DEER-refocused echo intensity (grey lines) is plotted versus evolution time for each spin-labeled position at pH 7.6 (resting state) and pH 4.6 (desensitized state) and fit using model-free Tikhonov regularization (pH 7.6, black lines; pH 4.6, blue lines). Pairs of data were recorded on the same spectrometers and under identical conditions. (Right panels) The corresponding interspin distance distributions are plotted at pH 7.6 (black) and pH 4.6 (blue) with the mean distances for each peak labeled. For T157R1 and K247R1 samples at pH 4.6, we also collected data out to the same dipolar evolution times as the pH 7.6 samples (dotted blue lines).</p

Purified GLIC reconstituted into liposomes is functional.

<p>(A) Single-channel currents of purified GLIC C26A mutant protein reconstituted into PE∶PG, PE∶PG∶cholesterol, and PE∶PG∶cardiolipin liposomes were recorded in planar lipid bilayers composed of the same lipids. Representative single-channel current traces (left), and current-voltage relationships with single-channel conductance values (right) are shown. Open-dwell times τ_o for the GLIC C26A mutant were 11.83±0.06 ms when reconstituted into PE∶PG liposomes, 10.15±0.08 ms into PE∶PG∶cholesterol liposomes, and 19.64±0.06 ms into PE∶PG∶cardiolipin liposomes, respectively. (B) Single-channel currents of purified GLIC mutants (K32C, T157C, and P249C) reconstituted into PE∶PG liposomes were recorded in planar lipid bilayers composed of the same lipids. Representative single-channel current traces (left) and current-voltage relationships with single-channel conductance values (right) are shown. (C) Currents induced by pH jumps from uninjected <i>Xenopus laevis</i> oocytes and ooctyes injected with purified, single cysteine MTSL-labeled GLIC protein reconstituted into PE∶PG liposomes (C26A, K32C, T157C, K247C, and P249C). Currents from GLIC-protein injected oocytes were significantly larger than those from uninjected oocytes.</p

Proton-induced changes in spin probe mobility, ΔH₀⁻¹.

<p>For each GLIC mutant (C26R1, K32R1, T157R1, K247R1, and P249R1), the inverse width of the central line of the CW spectra, ΔH₀⁻¹, is plotted at pH 7.6 (black), pH 4.6 (blue), and pH 3.0 (red, K247R1 and P249R1 only). An increase in ΔH₀⁻¹ reflects increased R1 mobility, whereas a decrease reflects decreased mobility.</p