Higher Susceptibility to Halothane Modulation in Open- Than in Closed-Channel α4β2 nAChR Revealed by Molecular Dynamics Simulations

Abstract

The neuronal α4β2 nicotinic acetylcholine receptor (nAChR) is a potential molecular target for general anesthetics. It is unclear, however, whether anesthetic action produces the same effect on the open and closed channels. Computations parallel to our previous open channel study (J. Phys. Chem. B, 2009) were performed on the closed-channel α4β2 nAChR to investigate the conformationdependent anesthetic effects on channel structures and dynamics. Flexible ligand docking and over 20-ns molecular dynamics simulations revealed similar halothane-binding sites in the closed and open channels. The sites with relatively high binding affinities (~−6.0 kcal/mol) were identified at the interface of extracellular (EC) and transmembrane (TM) domains or at the interface between α4 and β2 subunits. Despite similar sites for halothane binding, the closed-channel conformation showed much less sensitivity than the open channel to the structural and dynamical perturbations from halothane. Compared to the systems without anesthetics, the amount of water inside the pore decreased by 22% in the presence of halothane in the open channel, but only by 6% in the closed channel. Comparison of the non-bonded interactions at the EC/TM interfaces suggested that the β2 subunits were more prone than the α4 subunits to halothane binding. In addition, our data support the notion that halothane exerts its effect by disturbing the quaternary structure and dynamics of the channel. The study concludes that sensitivity and global dynamics responsiveness of α4β2 nAChR to halothane are conformation dependent. The effect of halothane on global dynamics of openchannel conformation might also account for the action of other inhaled general anesthetics.

Multiple binding sites for the general anesthetic isoflurane identified in the nicotinic acetylcholine receptor transmembrane domain

“…Moreover, residues in the GABA A and glycine receptors have been identified that contribute to positive modulation of those receptors, using photolabeling and mutagenesis (1,3,(19)(20)(21)(22)(23)(24); some of these residues are thought to lie at the subunit interfaces in a site analogous to the intersubunit sites we observe, according to experimentally confirmed (34) homology models based on the nAChR. A similar site is occupied in simulations of halothane interacting with the nAChR (30,35). The site is highly suggestive as an allosteric modulation site, as the M2-M3 loop is thought to act in transduction of a ligand binding signal to the pore (12).…”

Seeking Structural Specificity: Direct Modulation of Pentameric Ligand-Gated Ion Channels by Alcohols and General Anesthetics

“…Simulations of nicotinic receptors based on the low-resolution Torpedo structures revealed conformation-dependent binding of the volatile anesthetics halothane and isoflurane that could influence domain interactions or occlude the pore (Vemparala et al, 2006;Brannigan et al, 2010;Liu et al, 2010). More recently, the characterization of both atomicresolution structure and anesthetic sensitivity in GLIC provided valuable new simulation templates.…”

Pentameric ligand-gated ion channels: insights from computation

“…Automated docking has been used to provide initial coordinates for further unrestrained simulations[43, 85, 86], which has primarily indicated sites on the surface of the protein or at the interface between the ECD and TMD. The alternative flooding approach[87, 88] begins the simulation with a (usually high) concentration of the anesthetic dispersed randomly in the water and allows it to partition into the membrane and protein sites.…”

“…Although automated docking methods cannot typically be used to measure an absolute binding affinity, Bertaccini et al[90] demonstrated that automated docking scores of propofol analogs display log linear correlation with EC 50 s for GABAAr potentiation, indicating that such scores can be used to estimate relative a nities of similar compounds. Liu et al [85, 86] used AFEP to estimate absolute binding a nities of halothane for both open and closed conformations of an α 4 β 2 model of a neuronal nAChR, finding that shallow binding sites tested were low-affinity but a deeper intersubunit site indicated by experiments was moderate to high affinity. AFEP calculations of isoflurane and propofol binding to two locations in GLIC (Figure 4), the pore and the allosteric intrasubunit site indicated by crystal structures, predicted that isoflurane has higher affinity for the pore while propofol has similar affinity for the pore and intrasubunit site.…”