Isoflurane alters the structure and dynamics of GLIC

Abstract

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.

Figure 1

Isoflurane binding sites in system X and system Y at the beginning and end of each simulation. (a and b) are top views of systems X and Y at the beginning of each simulation, respectively. (c and e) and (d and f) are top and side views at the end of each simulation for systems X and Y, respectively. Subunit labels (gray letters) and colors are consistent throughout all figures in the manuscript. Isoflurane molecules are colored according to their final binding sites: yellow for those in the intersubunit sites in the TM domain; green for those in intrasubunit sites in the TM domain; red for those at or close to the EC/TM interface; purple for the one inside the pore; and orange for those in the EC domain.

Figure 2

Stacked bar graphs representing the probability of the salt bridge occurrence between residues of loop C in the principal subunit and residues in the adjacent complementary subunit over the course of each simulation: (a) control system, (b) system X, and (c) system Y. Subunits for salt bridges at the interface are labeled accordingly. Number of simultaneous salt bridges in a single frame is indicated by single hash marks, a single salt bridge; double hash marks, two salt bridges; solid bar, three salt bridges. Salt bridges were examined in frames collected every 20 ps using the saltbr plugin of VMD (32). Salt bridges were considered as formed if the distance between the center of mass of the oxygen atoms in the acidic side chain and center of mass of the nitrogen atoms in the basic side chain was <4 Å.

Figure 3

Polar plots of radial (θ) and lateral (δ) tilt angles of the TM2 helices for (a) control; (b) system X; and (c) system Y. The θ angle defines the inclination of the principal axis of a TM2 helix with respect to the direction of the channel symmetry axis. The δ angle measures the orientation of the principal axis of a TM2 helix projected onto a lateral plane that is perpendicular to the radial plane. Every 25-ns simulation trajectory is colored differently in a time order of black, red, green, blue, and magenta (last 2 ns). The averaged lateral angels for the first and last 5-ns simulations are marked by black ticks. The principal axis of each TM2 helix was calculated based on coordinates of backbone atoms of residues E222–I240.

Figure 4

Mean squared fluctuation of the control system (black), system X (purple), and system Y (green) based on three slowest modes of GNM analysis (39,40). Structures after 102-ns simulations were used for GNM analysis. Each residue of GLIC within 3.5 Å of isoflurane for >30 ns in the last 40-ns simulation is marked with a diamond (system X) or a circle (system Y) that are colored the same as isoflurane molecules at each equivalent site shown in Fig. 1. Gray triangles indicate initial isoflurane positions where isoflurane migrated away from the protein during the simulations. The GLIC secondary structure is highlighted at the top of the figure.

Figure 5

Correlated motions of GLIC. (a) Cross correlation map of GLIC in the control simulation cMD1 reveals residues within a subunit or between subunits that fluctuate in the same direction at the same time. (b) Cross correlation map of GLIC in the xMD1 simulation of the X system. (c) Cross correlation map of GLIC in the yMD1 simulation of the Y system. Note those motions that exist in b and c, but not in a: the strong anticorrelated motions in EC domains between different subunits and the strong correlated motions among TM4, TM3, and TM1. The color scale runs from blue (anticorrelated motion) to red (correlated motion).