Molecular modeling of a tandem two pore domain potassium channel reveals a putative binding Site for general anesthetics

[Image: see text] Anesthetics are thought to mediate a portion of their activity via binding to and modulation of potassium channels. In particular, tandem pore potassium channels (K2P) are transmembrane ion channels whose current is modulated by the presence of general anesthetics and whose genetic absence has been shown to confer a level of anesthetic resistance. While the exact molecular structure of all K2P forms remains unknown, significant progress has been made toward understanding their structure and interactions with anesthetics via the methods of molecular modeling, coupled with the recently released higher resolution structures of homologous potassium channels to act as templates. Such models reveal the convergence of amino acid regions that are known to modulate anesthetic activity onto a common three- dimensional cavity that forms a putative anesthetic binding site. The model successfully predicts additional important residues that are also involved in the putative binding site as validated by the results of suggested experimental mutations. Such a model can now be used to further predict other amino acid residues that may be intimately involved in the target-based structure–activity relationships that are necessary for anesthetic binding

The Molecular Mechanisms of Anesthetic Action: Updates and Cutting Edge Developments from the Field of Molecular Modeling

For over 160 years, general anesthetics have been given for the relief of pain and suffering. While many theories of anesthetic action have been purported, it has become increasingly apparent that a significant molecular focus of anesthetic action lies within the family of ligand-gated ion channels (LGIC’s). These protein channels have a transmembrane region that is composed of a pentamer of four helix bundles, symmetrically arranged around a central pore for ion passage. While initial and some current models suggest a possible cavity for binding within this four helix bundle, newer calculations postulate that the actual cavity for anesthetic binding may exist between four helix bundles. In either scenario, these cavities have a transmembrane mode of access and may be partially bordered by lipid moieties. Their physicochemical nature is amphiphilic. Anesthetic binding may alter the overall motion of a ligand-gated ion channel by a “foot-in-door” motif, resulting in the higher likelihood of and greater time spent in a specific channel state. The overall gating motion of these channels is consistent with that shown in normal mode analyses carried out both in vacuo as well as in explicitly hydrated lipid bilayer models. Molecular docking and large scale molecular dynamics calculations may now begin to show a more exact mode by which anesthetic molecules actually localize themselves and bind to specific protein sites within LGIC’s, making the design of future improvements to anesthetic ligands a more realizable possibility

The Molecular Mechanisms of Anesthetic Action: Updates and Cutting Edge Developments from the Field of Molecular Modeling

pharmaceutical

Molecular Mechanism for the Dual Alcohol Modulation of Cys-loop Receptors

Samuel Murail is with Stockholm University, Rebecca J. Howard is with UT Austin, Torben Broemstrup is with Stockholm University, Edward J. Bertaccini is with the Palo Alto Veterans Affairs Health Care System and Stanford University School of Medicine, R. Adron Harris is with UT Austin, James R. Trudell is with Stanford University School of Medicine, Erik Lindahl is with Stockholm University.Cys-loop receptors constitute a superfamily of pentameric ligand-gated ion channels (pLGICs), including receptors for acetylcholine, serotonin, glycine and γ-aminobutyric acid. Several bacterial homologues have been identified that are excellent models for understanding allosteric binding of alcohols and anesthetics in human Cys-loop receptors. Recently, we showed that a single point mutation on a prokaryotic homologue (GLIC) could transform it from a channel weakly potentiated by ethanol into a highly ethanol-sensitive channel. Here, we have employed molecular simulations to study ethanol binding to GLIC, and to elucidate the role of the ethanol-enhancing mutation in GLIC modulation. By performing 1-µs simulations with and without ethanol on wild-type and mutated GLIC, we observed spontaneous binding in both intra-subunit and inter-subunit transmembrane cavities. In contrast to the glycine receptor GlyR, in which we previously observed ethanol binding primarily in an inter-subunit cavity, ethanol primarily occupied an intra-subunit cavity in wild-type GLIC. However, the highly ethanol-sensitive GLIC mutation significantly enhanced ethanol binding in the inter-subunit cavity. These results demonstrate dramatic effects of the F(14′)A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation.This work was supported by grants from the Swedish Research Council (2010-491,2010-5107), the European Research Council (209825), the Swedish Foundation for Strategic Research, the Swedish e-Science Research Center, National Institutes of Health/National Institutes on Alcohol Abuse and Alcoholism Grants T32 AA007471, R01 AA06399, and R01 AA013378. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Waggoner Center for Alcohol and Addiction Researc

Insights into the Nature of Anesthetic–Protein Interactions: An ONIOM Study

Anesthetics have been employed widely to relieve surgical suffering, but their mechanism of action is not yet clear. For over a century, the mechanism of anesthesia was previously thought to be via lipid bilayer interactions. In the present work, a rigorous three-layer ONIOM­(M06-2X/6-31+G*:PM6:AMBER) method was utilized to investigate the nature of interactions between several anesthetics and actual protein binding sites. According to the calculated structural features, interaction energies, atomic charges, and electrostatic potential surfaces, the amphiphilic nature of anesthetic–protein interactions was demonstrated for both inhalational and injectable anesthetics. The existence of hydrogen and halogen bonding interactions between anesthetics and proteins was clearly identified, and these interactions served to assist ligand recognition and binding by the protein. Within all complexes of inhalational or injectable anesthetics, the polarization effects play a dominant role over the steric effects and induce a significant asymmetry in the otherwise symmetric atomic charge distributions of the free ligands <i>in vacuo</i>. This study provides new insight into the mechanism of action of general anesthetics in a more rigorous way than previously described. Future rational design of safer anesthetics for an aging and more physiologically vulnerable population will be predicated on this greater understanding of such specific interactions

Anesthetic Binding Site

The superfamily of ligand-gated ion channels (LGICs) has been implicated in anesthetic and alcohol responses. Mutations within glycine and GABA receptors have demonstrated that possible sites of anesthetic action exist within the transmembrane subunits of these receptors. The exact molecular arrangement of this transmembrane region remains at intermediate resolution with current experimental techniques. Homology modeling methods were therefore combined with experimental data to produce a more exact model of this region. A consensus from multiple bioinformatics techniques predicted the topology within the transmembrane domain of a glycine alpha one receptor (GlyRa1) to be alpha helical. This fold information was combined with sequence information using the SeqFold algorithm to search for modeling templates. Independently, the FoldMiner algorithm was used to search for templates that had structural folds similar to published coordinates of the homologous nAChR (1OED). Both SeqFold and Foldminer identified the same modeling template. The GlyRa1 sequence was aligned with this template using multiple scoring criteria. Refinement of the alignment closed gaps to produce agreement with labeling studies carried out on the homologous receptors of the superfamily. Structural assignment and refinement was achieved using Modeler. The final structure demonstrated a cavity within the core of a four-helix bundle. Residues known to be involved in modulating anesthetic potency converge on and line this cavity. This suggests that the binding sites fo