Anesthetic binding in a pentameric ligand-gated ion channel: GLIC

Abstract

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K(D)) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.

Figure 1

Three tryptophan-associated sites in the wild-type GLIC: W47 and W72 in the EC domain (termed as Site-Trp^EC), W160 at the EC-TM interfacial region (termed as Site-Trp^INT), and W213 and W217 in the TM domain (termed as Site-Trp™).

Figure 2

Fluorescence quenching of wild-type GLIC (square), GLIC^EC (circle), GLIC^INT (triangle), and GLIC™ (diamond) by various concentrations of (A) volatile anesthetic halothane and (B) intravenous anesthetic thiopental. (Solid lines) Nonlinear fitting of experimental data to Eq. 2. The experimental error bars (∼5%) are omitted for clarity. The maximum quenching Q_max and anesthetic disassociation constant K_D from fittings are presented in Table 1. The quenching of wild-type GLIC by potassium bromide (pentagon) was included in panel A as reference.

Figure 3

Fluorescence quenching of GLIC^N200W, a mutant of the anesthetic binding site predicted by computer docking, by (A) volatile anesthetic halothane and (B) intravenous anesthetics thiopental. The solid lines resulted from nonlinear fitting of the experimental data using Eq. 2. The error bars are the standard deviations of three experiments. The fitting results are included in Table 1.

Figure 4

Halothane molecules (van der Waals format) at putative binding sites near TRP residues (licorice format) of GLIC or N200W of the mutant, suggested by halothane docking on the MD-relaxed protein structures. (A) Near W47 and W72 in the EC domains of GLIC; (B) near W160 at the EC-TM regions; (C) near W213 and W217 in the TM domains; and (D) near N200W at the EC-TM regions of the mutant. Note that most of halothane molecules are bound to the pocket within a subunit. Bromide or chloride atom of halothane interacts directly with the indole ring of TRP at most sites.

Figure 5

Predicted binding for intravenous anesthetic thiopental near W160 at the pocket between two subunits colored in lime and orange. Docking was performed on a snapshot of GLIC after a 5-ns MD simulation in a fully hydrated POPG-POPE mixture. Only the docking with the lowest energy was shown. Thiopental is highlighted with mesh surfaces. Sulfur atom of thiopental is shown in a large yellow sphere. Oxygen, nitrogen, carbon, and hydrogen of anesthetics are shown in red, blue, tan, and white, respectively. Sulfur atom of thiopental interacts directly with the indole ring of TRP.

Figure 6

Putative binding of intravenous anesthetic thiopental inside the GLIC channel, predicted by docking on the MD relaxed GLIC structure. For clarity, only four TM2 subunits are shown. Residues S226, I233, and I240 are plotted in licorice format. Sulfur atom of thiopental is shown in a large sphere. Oxygen, nitrogen, carbon, and hydrogen of anesthitcs are colored differently. The docking energy of thiopental is −5.2 ± 0.3 Kcal/mol and the docking occupancy is 4%.

Figure 7

(A) Comparison of the subunit root-mean-squared-fluctuation (RMSF) of GLIC in the absence (black) and presence (red) of halothane near N200 over the last 1-ns simulation. (B) The ratio of RMSFs in the presence and absence of halothane is color-coded onto a subunit structure of GLIC. The regions having values significantly smaller (i.e., reducing RMSF) or greater (i.e., increasing RMSF) than one are colored in blue or red, respectively. The regions in white color had no significant change in RMSF.

Figure 8

(A) Salt bridge between D32 and R192 in the control system of GLIC. The salt bridge was perturbed by halothane binding near W160. (B) Halothane interacts with R192, and (C) halothane interacts with D32. Halothane molecules are shown in van der Waals format and protein residues are presented in licorice format.