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Graphical abstract
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Figure 1. Proposed Sites of General Anesthetic Modulation in the Pore and Upper TMD of Pentameric Ligand-Gated Ion Channels
(A) Cut-away view from the membrane plane (brown) of a pLGIC (gray) including the M2 helices (tan), with two proximal subunits transparent to reveal the pore (green). Putative propofol positions are shown as spheres (cyan).
(B) Simplified cross-sectional model of the GLIC TMD, viewed from the extracellular side, showing M2 helices (tan) in each of the five subunits. TMD helices (gray) defining putative anesthetic sites (cyan) associated with the upper right subunit are labeled (M1–M4).
See also Figure S1.
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Figure 2. Altering Electrostatic Contacts (N239C and H235Q) Enables Pore Opening by Anesthetics
(A) Left: electrostatic-contact residues N239 and H235 mapped in WT GLIC under activating conditions (PDB: 4HFI), viewed from the membrane plane. Center: zoom view from the pore shows the upper TMD of two subunits. Residues associated with general anesthetic modulation are shown as balls and sticks, colored by heteroatom (N, blue; O, red; and S, yellow). Right: contact model of GLIC TMD in the apparent open state is viewed from the extracellular side, with putative mediators of anesthetic effects shown for the upper right subunit. In all panels, residues of interest are colored according to the key (box) as follows: M205, dark red; H235, orange; N239, purple; E243, pink; S230, green; V242, tan; N200, dark gray; T255, gray; and Y197, light gray. Black lines indicate possible H-bonds.
(B) Electrophysiology data showing reduced pH sensitivity (left) and reversed modulation of EC10 currents by 30 μM propofol and 1 mM bromoform (right) in GLIC-N239C (purple) and H235Q (orange) relative to WT (black). Data represent mean ± SEM; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
(C) Representative apo structure of locally closed GLIC variants N239C and H235Q, shown in full (left), zoom (center), and contact model (right) views as in (A). Arrows in zoom and model views indicate channel opening.
(D) Views as in (A) of GLIC-N239C in the presence of bromoform. Mesh indicates bromine anomalous signal, contoured at 4 σ.
(E) Views as in (A) of GLIC-H235Q in the presence of propofol.
See also Figures S2–S4.
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Figure 3. Bulky Substitution in M1 (M205W) Stabilizes Open-State Intrasubunit Binding
(A) Electrophysiology data showing activation (inset) and modulation of EC10 currents by a range of propofol concentrations (left) and 1 mM bromoform (right) in GLIC-M205W (burgundy) relative to WT (black). Data represent mean ± SEM; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
(B) Left: GLIC-M205W TMD crystallized with propofol, viewed as in Figure 2A, showing M205W (burgundy) and propofol (cyan) associated with the right-hand distal subunit. Right: extracellular view of the bromoform complex shows the unmasked bromine anomalous map (cyan) contoured at 4 σ in all five subunits.
(C) Upper TMD of two GLIC-M205W subunits, viewed as in Figure 2A from the channel pore, with propofol and neighboring residues, including the mutated M205W, in ball-and-stick representation. For comparison, M1-M205 from the right subunit is superimposed from GLIC WT (PDB: 4HFI, arrow). Inset: equivalent views show WT (above) and M205W (below) structures, with propofol as spheres. Scale indicates B-factor color scheme.
See also Figures S2, S3, and S5.
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Figure 4. Potentiation Corresponds to Interfacial Binding in F238A/N239A Variant
(A) Left: GLIC-F238A viewed as in Figure 3B. Spheres indicate propofol (cyan) within the right distal subunit and F238A (brown) and N239 (purple) associated with the distal subunit interface. Center: zoom view shows two GLIC-F238A subunits, colored as in Figure 3C. Right: contact model for GLIC WT and F238A variants is shown.
(B) Views as in (A) of GLIC-F238A/N239A, with propofol at the subunit interface. Upward arrow indicates sensitivity to propofol potentiation.
See also Figures S2 and S3.
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Figure 5. Propofol Binds at the 6’ Level in a Closed State of the GLIC Pore
(A) Top: cut-away view of locally closed GLIC (2-22′ variant) from the membrane plane; colors indicate M2 helices (tan) and propofol (cyan). Bottom: contact map as in Figure 4A shows propofol interactions with five S230 residues (green) in the closed pore.
(B) Zoom view as in (A), showing domain crosslink (K33C-L246C, yellow) and propofol (cyan) in the pore.
(C) Summary of two-electrode voltage-clamp recordings in oocytes showing enhanced gating (top) and inhibition of EC10 currents by 30 μM propofol and 1 mM bromoform (bottom) in GLIC WT (black) and S230T (green) variants. Data represent mean ± SEM; ∗p < 0.05.
See also Figures S2 and S3.
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Figure 6. A Multi-site Mechanism for Bimodal Modulation of pLGICs
Top: protonation favors a conformational change from apparent closed (C) to open (O) states, associated with a tilt of the upper M2 helices away from the pore (arrows in C).
Bottom: holo states are classified by anesthetic binding in the channel pore (P), within (W) intrasubunit sites, or between (B) subunits. Left to right: binding of anesthetic (cyan) in the channel pore is favored in the closed state (CP), represented here by the crosslinked 2-22′ variant. Additional weak binding within each closed-state subunit (CPW) is documented in some crosslinked channels (Laurent et al., 2016). Open-state binding within each subunit (OW) is permitted in WT GLIC (Nury et al., 2011) and further stabilized in variants H235Q and M205W; additional, weaker binding in the open pore (OPW) is documented in variant N239C. Binding between open-state subunits (OB) is unfavorable for WT GLIC, though sterically allowed in GLIC-F238A (bromoform) and -F238A/N239A (propofol).
Each model shows the five M2 helices (tan) plus additional helices of the upper right subunit and neighboring interface (gray). As defined in the key, colored circles represent anesthetic binding sites and key contacts implicated in anesthetic modulation for one subunit interface. Solid and dashed lines represent possible H-bonds and hydrophobic contacts, respectively. For clarity, upper-TMD-binding sites are indicated for only one subunit, although binding in up to five equivalent sites is expected.
See also Figure S6.
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Figure S1. Sequence alignment of residues implicated in general anesthetic modulation in representative pLGICs (related to Figure 1). Alignment of transmembrane helices M1–M3 from representative H. sapiens glycine, GABAA,
nACh, and 5-HT3 receptors, the crystallized construct of C. elegans GluClα (GluClαx), E. crysanthemi ELIC, and G. violaceus GLIC (bold). Sequences above and below the dotted line are primarily associated with potentiation and inhibition, respectively, by general anesthetics (represented by cyan propofol molecule and corresponding arrows at right). Numbers below indicate mutated GLIC residues M205 (burgundy), S230 (green), H235 (orange), and N239 (purple), plus additional propofol contacts within (dark gray, tan) and between subunits
(light gray, brown) and in the channel pore (green).
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Figure S2. Distances between putative hydrogen bond partners associated with the apparent open state (related to Figures 2–5). (A) Left, mean distance between the backbone carbonyl O atom of each M1-N200 residue and a side-chain O atom of the nearest M2-E243 residue on the + face of the neighboring subunit. For structures in the apparent closed state (clustered at left), this putative interaction is disrupted (≥9.80 Å); for most structures in the apparent open state (clustered at right), the interaction does not differ significantly from wild-type (3.80±0.01 Å). Right, zoom view as in Figure 2A of the interface between two wild-type GLIC subunits in the apparent open state (PDB ID 4HFI), showing a representative N200––E243+ interaction (black); as in most open state structures, the interaction is bridged by a water molecule (red).
(B) Left, mean distance between the backbone carbonyl O atom of each M1-N200 residue and a terminal side-chain atom of the nearest M2-N239 residue on the + face of the neighboring subunit. For structures in the apparent closed state, this putative interaction is disrupted (≥7.89 Å); for most structures in the apparent open state, the interaction does not differ significantly from wild-type (3.92±0.01Å). Note that values are not shown for the N239C or F238A/N239A variants, as hydrogen bonds are not chemically permitted at this position. Right, zoom view as in A of a GLIC open-state subunit interface, showing a representative N200––N239+ interaction (black) bridged by a water molecule (red). (C) Left, mean distance between the backbone carbonyl O atom of each M3-I259 residue and a side-chain N atom of the nearest M2-H235 (or H235Q) residue in the same subunit. For structures in the apparent closed state, this distance is ≥4.62Å; for all open-state structures except H235Q (orange), the interaction is ≤3.81Å. Right, zoom view as in A of open-state GLIC, showing a representative H235–I259 interaction (black). In all graphs, colored bar indicate wild-type (black), N239C (purple), H235Q (orange), crosslinked (green), M205W (burgundy), F238A (tan), and F238A/N239A (brown) GLIC variants; patterns indicate crystallization under resting conditions (boxes) or in the presence of
propofol (horizontal stripes) or bromoform (vertical stripes). Bold labels indicate the structures from this work. Error bars represent s.e.m. over five subunits; asterisks indicate significance vs. wild-type under activating conditions (PDB ID 4HFI), Dunnet’s multiple comparison test, analysis of variance, *p<0.001, **p<0.0001.
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Figure S3. Classification of past and present GLIC structures as apparent closed or open states (related to Figures 2–5). (A) RMSD of transmembrane helix Cα atoms in various structures, compared to wild-type GLIC under activating conditions (PDB ID 4HFI). Structures clustered at left, classified in the apparent closed state, all deviate from the apparent open state by >1.54 Å; structures at right, classified as apparent open-state, fall within 0.55Å. (B) Illustration of open-state comparisons in A, with representative superpositions of the GLIC-N239C TMD in the apo state (left, lavender) and in the presence of bromoform (right,
purple) compared to the apparent open state (gray), viewed from the extracellular side. Apostate GLIC-N239C deviates from the open state, particularly in the pore-lining M2 helices. (C) Transmembrane helix RMSD measurements as in A, compared to GLIC crystallized under resting conditions (PDB ID 4NPQ chains A–E). Structures at left fall within 0.79 Å of the apparent closed state; those at right deviate by >1.38 Å. (D) Illustration of closed-state comparisons in C, showing superpositions of apo (left, lavender) and holo (right, purple) GLIC-N239C TMDs compared to the apparent closed state (gray), viewed from the extracellular side. Holo GLIC-N239C deviates from the closed state in the M2 region. (E) Minimum diameter at the hydrophobic gate (I233, 9’) in each channel pore. Structures at left are constricted to ≤4.6 Å; structures at right are expanded to ≥5.6 Å. (F) Model for pore diameter measurements in E, showing full-length apo (left, lavender) and holo (right, purple) GLIC-N239C, viewed from the membrane plane. Dotted line indicates the level of the 9’ hydrophobic gate (black). In all graphs, colored bars indicate wild-type (black), N239C (purple), H235Q (orange), crosslinked (green), M205W (burgundy), F238A (tan), and F238A/N239A (brown) GLIC variants; patterns indicate crystallization under resting conditions (boxes) or in the presence of propofol (horizontal stripes) or bromoform (vertical stripes). Bold labels indicate the structures from this work.
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Figure S4. Anomalous bromine maps and zoom views of anesthetic-potentiated GLIC variants (related to Figure 2). (A) Extracellular views of the TMD of GLIC variant N239C in the absence (left) and presence (right) of bromoform, showing the mutated N239C residue (purple) and unmasked bromine anomalous map (cyan) contoured at 4σ. Note expansion of the channel pore (tan) upon bromoform binding. (B) Views as in A of GLIC-H235Q, showing the mutated H235Q residue (orange) in the absence (left) and presence (right) of bromoform.
(C) Zoom views of GLIC-H235Q in the absence (left) and presence (right) of bromoform, shown as in Figure 2D from the channel pore. Arrows in apo structure (left) indicate motion towards the apparent open state (right) upon anesthetic binding. Mesh indicates bromine anomalous signal, contoured at 4σ.
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Figure S5. Zoom view with anomalous bromine map for bromoform co-crystal structure of GLIC-M205W (related to Figure 3). View as in Figure 3B of GLIC-M205W co-crystallized with bromoform, showing bromoform (cyan) and neighboring residues, plus superimposed M1 helix and residue M205 from wildtype GLIC for comparison (arrow). Mesh indicates bromine anomalous signal, contoured at 4 σ. Inset, equivalent views of wild-type (above) and M205W (below) structures, with bromoform shown as spheres. Scale indicates occupancy shading scheme.
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Figure S6. Crystallographic landmarks and perturbations to an allosteric mechanism for general anesthetic modulation in GLIC (related to Figure 6).
Simplified mechanisms representing (A) wild-type, (B) crosslinked (e.g. 2-22’), (C) F238A/N239A, (D) H235Q, (E) N239C, and (F) M205W GLIC variants. Individual states are distinguished as in Figure 6 as apparent closed (C) or open (O), with anesthetics bound in the channel pore (P), within intrasubunit sites (W), or between transmembrane subunits (B). In each mechanism, cyan spheres represent general anesthetic binding, and pentagons indicate the presence of representative X-ray structures; solid pentagons indicate structures reported
in this work. For GLIC mutants (B–F), the canonical mechanism is rendered semitransparent; bold and dotted circles represent preferentially stabilized and destabilized states, respectively.
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