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PLoS Biol
2013 Nov 01;1111:e1001714. doi: 10.1371/journal.pbio.1001714.
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Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions.
Dellisanti CD
,
Ghosh B
,
Hanson SM
,
Raspanti JM
,
Grant VA
,
Diarra GM
,
Schuh AM
,
Satyshur K
,
Klug CS
,
Czajkowski C
.
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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.
Figure 2. Purified GLIC reconstituted into liposomes is functional.(A) Single-channel currents of purified GLIC C26A mutant protein reconstituted into PEâ¶PG, PEâ¶PGâ¶cholesterol, and PEâ¶PGâ¶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 Ïo for the GLIC C26A mutant were 11.83±0.06 ms when reconstituted into PEâ¶PG liposomes, 10.15±0.08 ms into PEâ¶PGâ¶cholesterol liposomes, and 19.64±0.06 ms into PEâ¶PGâ¶cardiolipin liposomes, respectively. (B) Single-channel currents of purified GLIC mutants (K32C, T157C, and P249C) reconstituted into PEâ¶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 Xenopus laevis oocytes and ooctyes injected with purified, single cysteine MTSL-labeled GLIC protein reconstituted into PEâ¶PG liposomes (C26A, K32C, T157C, K247C, and P249C). Currents from GLIC-protein injected oocytes were significantly larger than those from uninjected oocytes.
Figure 3. CW EPR spectra reveal proton-induced gating movements.Comparison of X-band CW EPR spectra of spin-labeled GLIC wild-type and mutant protein at pH 7.6 (black, closed state) and pH 4.6 (blue, desensitized state). Spectra were recorded at room temperature over 100 G. Pairs of data were recorded on the same spectrometers and under identical conditions. Immobile and mobile components in the low-field region of the K32R1 spectrum are indicated by arrows. The low-field regions of the K32R1, T157R1, and K247R1 spectra are enlarged to highlight the proton-induced changes. (Top left) Close-up view of GLIC crystal structure with spin-labeled positions C26, K32, T157, K247, and P249 shown in space-fill.
Figure 4. Proton-induced changes in spin probe mobility, ÎH0â1.For each GLIC mutant (C26R1, K32R1, T157R1, K247R1, and P249R1), the inverse width of the central line of the CW spectra, ÎH0â1, is plotted at pH 7.6 (black), pH 4.6 (blue), and pH 3.0 (red, K247R1 and P249R1 only). An increase in ÎH0â1 reflects increased R1 mobility, whereas a decrease reflects decreased mobility.
Figure 5. Effects of lipids on proton-induced gating motions.X-band CW EPR spectra of T157R1 reconstituted into PEâ¶PG (top), PEâ¶PGâ¶cholesterol (middle), and PEâ¶PGâ¶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 PEâ¶PG, whereas cardiolipin hindered gating-induced changes in T157R1 mobility.
Figure 6. Proton-induced distance changes revealed by DEER spectroscopy.(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).
Figure 7. Detergent prevents proton-induced GLIC gating motions.(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.
Figure 1. Location of loop 2, loop 9, and M2âM3 loop in GLIC.(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 M2âM3 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 M2âM3 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).
Altenbach,
High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation.
2008, Pubmed
Altenbach,
High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation.
2008,
Pubmed
Asmar-Rovira,
Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments.
2008,
Pubmed
Baenziger,
3D structure and allosteric modulation of the transmembrane domain of pentameric ligand-gated ion channels.
2011,
Pubmed
Barrantes,
Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review).
2002,
Pubmed
Bocquet,
X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.
2009,
Pubmed
Bocquet,
A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family.
2007,
Pubmed
,
Xenbase
Cieslak,
Electron spin-echo envelope modulation (ESEEM) reveals water and phosphate interactions with the KcsA potassium channel.
2010,
Pubmed
Criado,
Effects of lipids on acetylcholine receptor. Essential need of cholesterol for maintenance of agonist-induced state transitions in lipid vesicles.
1982,
Pubmed
Cuello,
Structural basis for the coupling between activation and inactivation gates in K(+) channels.
2010,
Pubmed
daCosta,
A lipid-dependent uncoupled conformation of the acetylcholine receptor.
2009,
Pubmed
daCosta,
Anionic lipids allosterically modulate multiple nicotinic acetylcholine receptor conformational equilibria.
2009,
Pubmed
daCosta,
Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor.
2004,
Pubmed
daCosta,
Lipid-protein interactions at the nicotinic acetylcholine receptor. A functional coupling between nicotinic receptors and phosphatidic acid-containing lipid bilayers.
2002,
Pubmed
daCosta,
Gating of pentameric ligand-gated ion channels: structural insights and ambiguities.
2013,
Pubmed
Dalziel,
The effect of cholesterol on agonist-induced flux in reconstituted acetylcholine receptor vesicles.
1980,
Pubmed
Dellisanti,
Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.
2007,
Pubmed
Fernandez-Ballester,
A role for cholesterol as a structural effector of the nicotinic acetylcholine receptor.
1994,
Pubmed
Ghimire,
Significantly improved sensitivity of Q-band PELDOR/DEER experiments relative to X-band is observed in measuring the intercoil distance of a leucine zipper motif peptide (GCN4-LZ).
2009,
Pubmed
Ghosh,
Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.
2013,
Pubmed
,
Xenbase
Gonzalez-Gutierrez,
Bridging the gap between structural models of nicotinic receptor superfamily ion channels and their corresponding functional states.
2010,
Pubmed
Hanson,
Structural mechanisms underlying benzodiazepine modulation of the GABA(A) receptor.
2008,
Pubmed
,
Xenbase
Hatmal,
Computer modeling of nitroxide spin labels on proteins.
2012,
Pubmed
Hibbs,
Principles of activation and permeation in an anion-selective Cys-loop receptor.
2011,
Pubmed
Hilf,
A prokaryotic perspective on pentameric ligand-gated ion channel structure.
2009,
Pubmed
Hilf,
X-ray structure of a prokaryotic pentameric ligand-gated ion channel.
2008,
Pubmed
Hilf,
Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.
2009,
Pubmed
Hubbell,
Recent advances in site-directed spin labeling of proteins.
1998,
Pubmed
Hubbell,
Watching proteins move using site-directed spin labeling.
1996,
Pubmed
Hubbell,
Identifying conformational changes with site-directed spin labeling.
2000,
Pubmed
Jarecki,
Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes.
2013,
Pubmed
,
Xenbase
Jha,
Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker.
2007,
Pubmed
Khatri,
Structural rearrangements in loop F of the GABA receptor signal ligand binding, not channel activation.
2009,
Pubmed
,
Xenbase
Klug,
Methods and applications of site-directed spin labeling EPR spectroscopy.
2008,
Pubmed
Labriola,
Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment.
2013,
Pubmed
Langen,
Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure.
2000,
Pubmed
Lee,
Principal pathway coupling agonist binding to channel gating in nicotinic receptors.
2005,
Pubmed
Leite,
Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: electrophysiology-coordinated photochemistry and mass spectrometry.
2003,
Pubmed
Li,
Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.
2011,
Pubmed
Lummis,
Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.
2005,
Pubmed
Lyford,
Agonist-induced conformational changes in the extracellular domain of alpha 7 nicotinic acetylcholine receptors.
2003,
Pubmed
,
Xenbase
Marsal,
Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.
1995,
Pubmed
,
Xenbase
Miller,
Binding, activation and modulation of Cys-loop receptors.
2010,
Pubmed
Miyazawa,
Structure and gating mechanism of the acetylcholine receptor pore.
2003,
Pubmed
Mukhtasimova,
Nicotinic receptor transduction zone: invariant arginine couples to multiple electron-rich residues.
2013,
Pubmed
Murzyn,
Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane.
2005,
Pubmed
Newell,
The GABAA receptor alpha 1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating.
2003,
Pubmed
,
Xenbase
Pannier,
Dead-time free measurement of dipole-dipole interactions between electron spins.
2000,
Pubmed
Parikh,
Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility.
2011,
Pubmed
Perozo,
Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy.
1998,
Pubmed
Pless,
Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain.
2009,
Pubmed
,
Xenbase
Prevost,
A locally closed conformation of a bacterial pentameric proton-gated ion channel.
2012,
Pubmed
Sooksawate,
Effects of membrane cholesterol on the sensitivity of the GABA(A) receptor to GABA in acutely dissociated rat hippocampal neurones.
2001,
Pubmed
Thompson,
Mutagenesis and molecular modeling reveal the importance of the 5-HT3 receptor F-loop.
2006,
Pubmed
Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed
Unwin,
Gating movement of acetylcholine receptor caught by plunge-freezing.
2012,
Pubmed
Vásquez,
A structural mechanism for MscS gating in lipid bilayers.
2008,
Pubmed
Velisetty,
Desensitization mechanism in prokaryotic ligand-gated ion channel.
2012,
Pubmed
Velisetty,
Conformational transitions underlying pore opening and desensitization in membrane-embedded Gloeobacter violaceus ligand-gated ion channel (GLIC).
2012,
Pubmed
Venkatachalan,
Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus.
2007,
Pubmed
,
Xenbase
Xiu,
A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors.
2005,
Pubmed
,
Xenbase
Yamodo,
Conformational changes in the nicotinic acetylcholine receptor during gating and desensitization.
2010,
Pubmed
Zhang,
Agonist- and antagonist-induced conformational changes of loop F and their contributions to the rho1 GABA receptor function.
2009,
Pubmed
,
Xenbase
Zou,
Increased sensitivity and extended range of distance measurements in spin-labeled membrane proteins: Q-band double electron-electron resonance and nanoscale bilayers.
2010,
Pubmed
