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FIGURE 1. Modulation of ELIC by bromoform.
a, chemical formulas of chloroform and bromoform. b, ELIC current traces were evoked by application of 5 mm GABA in the absence and presence of 200 μm bromoform or 100 mm bromoethanol. c, concentration-inhibition curves from the experiments shown in panel b. Squares are data for bromoform, and circles are data for bromoethanol (mean ± S.E.). Each data point represents the average of 3–10 experiments.
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FIGURE 2. X-ray crystal structure of ELIC in complex with bromoform.
a, side view of ELIC in graphic representation and overlay with anomalous electron density, shown as magenta mesh at a contour level of 4σ. Each subunit of the pentamer is shown in a different color. Anomalous densities can be observed in the extracellular domain, transmembrane domain, and channel pore. b and c, extracellular and intracellular view of ELIC along the five-fold symmetry axis. The pore site is lined by the M2-helices of each of the five subunits. The transmembrane site is formed at the interface between two subunits, involving the M1- and M4-helix of one subunit and the M3-helix of the neighboring subunit. Bromoform molecules are represented as a single magenta sphere. d, comparison of the bromoform binding site in the closed channel pore of ELIC (left) and bromo-lidocaine binding site in the open channel pore of GLIC (right). Bromoform and bromo-lidocaine occupy overlapping binding sites, which are located in the hydrophobic part of the ion conduction pathway (hydrophobic residues are colored in yellow, hydrophilic residues are in green, and charged residues are in red). e, mutagenesis experiments in ELIC demonstrate that the inhibitory effect of 200 μm bromoform is almost completely eliminated in L9′S and strongly reduced in F16′S mutants. The inhibitory effect of bromoform was tested at an EC20 concentration of GABA, which was 0.5 mm for L9′S and 3 mm for wild type and F16′S. Error bars represent mean ± S.E. from 3–5 different experiments. Asterisks indicate significant difference from wild type (p < 0.05).
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FIGURE 3. Bromoform stabilizes the ELIC pore in a closed conformation.
a, ribbon representation of the pore-lining M2-helix in the apoELIC structure (blue, PDB code 2vl0) and the bromoform-bound structure (red). The view is along the five-fold symmetry axis looking down on the channel pore from the extracellular domain. The dashed lines are distance measurements between 13′A Cα atoms of different subunits. b, pore radius analysis for apoELIC (blue), bromoform-bound ELIC (red), and GLIC, which likely corresponds to an open pore conformation (PDB code 3eam).
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FIGURE 4. Molecular recognition of bromoform at an intersubunit transmembrane site.
a and b, Comparison of the propofol binding site in GLIC (a) and the bromoform binding site of ELIC (b) in a surface representation. Propofol binds in an intrasubunit pocket in the upper half of the transmembrane domain. Bromoform binds in an intersubunit pocket farther down the transmembrane domain. The transmembrane domains of two neighboring subunits are shown in orange and blue. The propofol and bromoform pockets are highlighted in yellow. The extracellular domain is shown in white. The inset shows a more detailed view of the intersubunit transmembrane site. The magenta mesh represents anomalous electron density contoured at 4σ. c, cross-section through a surface representation of ELIC. The intersubunit bromoform site is formed at a pre-existing cavity, which is occupied by a bromoform molecule (single bromine atoms are shown as magenta spheres) in all five sites. d, detailed view of the amino acid residues in ELIC that form the intersubunit bromoform site (highlighted in yellow). The site is formed at the interface between M1 and M4 of one subunit (blue) and M3 of the neighboring subunit (orange).
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FIGURE 5. Quenching of intrinsic tryptophan fluorescence in ELIC by bromoform. Increasing concentrations of bromoform cause quenching of intrinsic fluorescence in ELIC. This effect is strongly reduced in the double mutant W221Y/W225Y. Each data point is the average of 3 experiments. Error bars indicate S.E.
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FIGURE 6. Conservation of bromoform binding residues at a transmembrane intersubunit site in pLGICs. Alignment of ELIC, GLIC, and selected sequences of human GlyR, GABAA/CR, nAChR, and 5-HT3Rs is shown. Numbers at the top of the alignment correspond to ELIC residues, and numbers at the bottom correspond to α1 GlyR residues. Residues are colored in shades of blue using an identity threshold of 50%. Yellow residues correspond to the highlighted residues in the intersubunit bromoform site in ELIC (Fig. 3d). Aromatic residues at these positions as well as Pro-307 are strongly conserved in GlyR and GABAA/CR, but not in nAChR and 5-HT3Rs. The dashed line separates inhibitory (top) from excitatory (bottom) receptors.
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FIGURE 7. Mutagenesis of the intersubunit bromoform binding site in the human α1 glycine receptor.
a, graphic representation of the intersubunit bromoform binding site in the transmembrane domain of ELIC. Interacting residues are shown in yellow sticks. The bromoform molecule is shown as a single magenta sphere. b, two-electrode voltage clamp recordings from oocytes expressing wild type α1 GlyRs, F295A, and Y301A mutants. Each current trace is shown in a specific color to facilitate comparison of the range of glycine concentrations: 3 μm (green), 10 μm (red), 30 μm (blue), 100 μm (yellow), 300 μm (magenta), 1 mm (cyan), and 3 mm (black). To reach saturation for Tyr-301 receptors, we also applied 10 mm (orange). Each oocyte was exposed to the same range of glycine concentrations in the presence of 200 μm bromoform (BrF). The insets for WT and F295A show a magnified view of traces obtained at low glycine concentrations. The asterisk indicates potentiation by bromoform. c, concentration-activation curves for WT, F295A, and Y301A GlyRs in the absence of bromoform. d–f, concentration-activation curves in the absence (gray line, −BrF) and presence (black line, +BrF) of 200 μm bromoform for WT (d), F295A (e), and Y301A (f) receptors. Error bars in panels c–f indicate S.E.
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FIGURE 8. Overview of different general anesthetic binding sites revealed in crystal structures of pLGICs. A graphic representation of two neighboring subunits of the ELIC pentamer is shown. The different spheres correspond to different binding site for general anesthetics: the propofol-desflurane site (green) (18), the alcohol-ivermectin site (yellow) (14), and the three bromoform sites identified in this study (magenta). ES, extracellular site; PS, pore site; IS, intersubunit site).
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