Microsecond Simulations Indicate that Ethanol Binds between Subunits and Could Stabilize an Open-State Model of a Glycine Receptor

Abstract

Cys-loop receptors constitute a superfamily of ion channels gated by ligands such as acetylcholine, serotonin, glycine, and γ-aminobutyric acid. All of these receptors are thought to share structural characteristics, but due to high sequence variation and limited structure availability, our knowledge about allosteric binding sites is still limited. These sites are frequent targets of anesthetic and alcohol molecules, and are of high pharmacological importance. We used molecular simulations to study ethanol binding and equilibrium exchange for the homomeric α1 glycine receptor (GlyRα1), modeled on the structure of the Gloeobacter violaceus pentameric ligand-gated channel. Ethanol has a well-known potentiating effect and can be used in high concentrations. By performing two microsecond-scale simulations of GlyR with/without ethanol, we were able to observe spontaneous binding in cavities and equilibrium ligand exchange. Of interest, it appears that there are ethanol-binding sites both between and within the GlyR transmembrane subunits, with the intersubunit site having the highest occupancy and slowest exchange (∼200 ns). This model site involves several residues that were previously identified via mutations as being crucial for potentiation. Finally, ethanol appears to stabilize the GlyR model built on a presumably open form of the ligand-gated channel. This stabilization could help explain the effects of allosteric ligand binding in Cys-loop receptors.

Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

“…However, even relatively long time-scale molecular dynamics simulations (1-s simulation of ethanol binding to GlyR with a large Linux cluster and 6 weeks ϫ 120 of computer time) (Murail et al, 2011) of GlyR in the presence or absence of ethanol do not show substantial changes in the tertiary structure of GlyR.…”

“…Loop 2 in GLIC is essentially identical to the corresponding loop 2 in the X-ray structure of the glutamate chloride channel (Hibbs and Gouaux, 2011). We chose to prepare a homology model based on GLIC because long time-scale molecular dynamics studies in GLIC (Nury et al, 2010(Nury et al, , 2011Zhu and Hummer, 2010) have allowed us to interpret interactions of salt bridges between the ligand-binding extracellular and TM domains of GlyR (Kash et al, 2003) in terms of dynamic interactions on the microsecond time scale (Murail et al, 2011).…”

Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

“…S4); methyl MTS modification of either residue could orient the methanethiolate label similarly to an alcohol molecule. Recent simulations of the closely related GlyR indicate that occupation of this cavity could stabilize the open state of the channel (40). Conversely, other regions of the protein-including the intrasubunit cavity that was recently implicated in inhibition by volatile anesthetics (32)-were relatively unaltered in the F(14′)A mutant ( Fig.…”

“…2C). Indeed, a cavity homologous to the intersubunit-linking tunnel region was occupied by ethanol in a recent 2-μs molecular dynamics simulation in the α1 GlyR (40).…”

A Molecular Mechanism of Ethanol Dependence: The Influence of the Ionotropic Glutamate Receptor Activated by N-Methyl-D-Aspartate

“…The main reason for a shift towards the protein theory originates from evidence that alcohol, at concentrations in the 10-20 mM range, directly interferes with the function of several ion channels (K + , Ca 2+ ) and receptors (Lovinger et al, 1989). These ethanol effects are mediated through a number of neural transmitter systems including γ-aminobutyric acid (GABA) and glutamate (Takadera et al, 2008;Murail et al, 2011).…”