Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State

Abstract

Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates.

Keywords: anesthesia; barbiturates; crystallography; electrophysiology; ligand-gated ion channels; membrane protein; structural biology; x-ray crystallography.

FIGURE 1.

Summary of the different steps in the chemical synthesis of selenocyanobarbital (A) and thiopental (B). DMF, Dimethylformamide; OTHP, O Tetrahydropyran; PTSA, p-Toluenesulfonic acid.

FIGURE 2.

The effect of barbiturates on the proton-induced currents of GLIC expressed in Xenopus laevis oocytes. A, concentration-inhibition curves for pentobarbital, bromobarbital, selenocyanobarbital, and thiopental on wild-type GLIC currents activated at pH 5.5. Data points are means ± S.E. for n = 5–6 oocytes. B–E, representative currents evoked by pH 5.5 showing inhibition of GLIC activation in the presence of 300 μm pentobarbital (B), 300 μm bromobarbital (C), 100 μm selenocyanobarbital (D), or 30 μm thiopental (E). F, representative proton current revealing a rebound current after washout of 300 μm thiopental. Dashed line indicates the amplitude of pH 5.5 evoked response in the absence of thiopental.

FIGURE 3.

Side view of the full structure of GLIC showing the barbiturate binding site. M2 helices are represented as gray ribbons. The box denotes the location of the observed barbiturate binding site (represented in yellow). This site overlaps with the previously described xenon (28) and bromoform (29) binding sites in GLIC.

FIGURE 4.

The observed binding sites of different barbiturates within the central ion channel pore of locally closed GLIC. A, structures of the compounds found to bind the central ion channel pore of GLIC. B, bromobarbital. C, thiopental. D, selenocyanobarbiturate. Note the inversion of thiopental when compared with bromo- and selenocyanobarbiturate. E and F, generated symmetric selenocyanobarbiturates. E, side view, F, top view. Residues of interest and ligands are shown as sticks, with carbons of hydrophobic residues colored beige; polar residues colored light blue; and ligands colored yellow. For heteroatoms: sulfur is colored green; bromine is orange; selenium is purple. Density maps were calculated from final refined structures, 2F_o − F_c was contoured at 1 σ level and carved around the ligand, and anomalous peaks are shown at 6 σ and 7 σ levels for bromobarbital and both selenocyanobarbiturate and thiopental, respectively.