General anesthetics, despite differences among them at the molecular level, modulate ligand-gated ion channels in a highly generalized fashion. A coarse-grained simulation model of this behavior now offers valuable insights into common molecular mechanisms of anesthetic action.
Joshua Mincer, MD, PhD, assistant professor, and Thomas T. Joseph, MD, PhD, CA-2 resident and an Eliasberg Research Scholar, both from the Department of Anesthesiology at Mount Sinai Hospital, New York City, presented their research at the most recent Anesthesiology Residents’ Night, held at the New York Academy of Medicine. Their work won first prize and applause from the anesthesiologists in attendance.
Scientists recognized early on that a nonspecific mechanism of anesthetic action could account for the fact that diverse molecules could cause general anesthesia. An earlier unifying theory of anesthesia, called the Meyer-Overton principle, highlighted the observation that the more lipid-soluble a molecule was, the more potent the anesthetic effect. This led to the idea that anesthetics imparted their effect by dissolving in the lipid membrane of neurons and interfering with their function in an otherwise nonspecific manner.
Although the lipid membrane persists in some modern theories of anesthesia, ion channels are now a central part of most theories. “What an ion channel does is help modulate the relative concentrations of ions inside and outside the cell,” Dr. Joseph explained. “Ion channels are in the cell membrane, regulating the ion concentrations on either side, and since ions are charged they are also regulating the electrical potentials on either side. That is the basis through which, for example, neuronal action potentials are propagated.” One could therefore theorize that disrupting the function of these channels might then disrupt brain function. The challenge, then, is to understand how general anesthetics, which are molecularly diverse, target and modulate these ligand-gated ion channels (LGICs).
Drs. Joseph and Mincer created their model by employing molecular dynamics, which traditionally takes into account every atom that is present in a particular system. “In this system, you have an ion channel, which is a very large protein with multiple subunits and is embedded in a lipid bilayer, which is comprised of a number of large molecules that are glommed together to form a cell membrane,” Dr. Joseph said.
The problem with creating a simulation that incorporates all the atoms in the system is that it takes a huge amount of computational power to get just a small amount of simulation time—often measured in nanoseconds. “In a given day you might produce 15 nanoseconds of simulation time, which is much shorter than most biological processes. In a coarse-grained model, however, you trade some of the fidelity of the full simulation to create longer time scales, and as long as you are careful about the conclusions you reach, you can learn new things that you couldn’t from a full, all-out model,” Dr. Joseph said. Both Drs. Joseph and Mincer used all-atom molecular dynamics in their respective dissertations (each has a PhD in computational/theoretical biophysics). Dr. Mincer has also done research in the use of coarse-grained methodology in order to create computer models that give results on a timescale large enough to be biologically relevant.
Besides yielding to more complex analysis, a simulation results in a trajectory, essentially a movie that can be viewed onscreen. The resultant images are faithful to reality. “It is not a cartoon,” explained Dr. Mincer. “Each of these images is calculated based on the laws of physics. So to the extent the physics approximations are right, it should mimic what is actually happening in vivo.”
Propofol and Xenon
The researchers started with a crystal structure of the Gloeobacter violaceous LGIC, a bacterial homolog to the nicotinic acetylcholine receptors that is considered to be a good structural model for human ion channels. A coarse-grained model of the LGIC from the bacterium was constructed. Propofol was first studied, partly as a validation to show that the coarse-grained model devised by the researchers matched up well with experimental results and with earlier all-atom studies of LGICs. “We needed to show that there was some connection between the two models,” Dr. Joseph explained. “And it turns out that even given the limitations of the coarse-grained model, it nonetheless shows things seen in the all-atom model.”
The researchers found that in the coarse-grained model, propofol preferentially localized to the correct area on LGIC—i.e., a site determined by crystallographic measurements to bind propofol. There is in fact evidence from prior research that multiple different anesthetic molecules can fit into that binding site, and so the researchers then looked at a coarse-grained model for xenon, which, being a noble gas, is chemically simpler than the other anesthetics, and found that the binding site also has affinity for it, which is a new finding. “We believe the ability of such a model to produce these results is consistent with a mechanism of general anesthetic function that requires minimal atomic-level selectivity,” Dr. Mincer said.
The research also looked at fluctuations within the structure. “If you consider what happens when you bind a ligand to one part of the ion channel, other parts get influenced, but they are farther away from the site of the binding, so you wonder how did that information travel from the binding site to the rest of the molecule,” Dr. Joseph said. “We analyzed the dynamics of our coarse-grained model to identify the residues in the protein that are most important in propagating that information, and showed that there are actually quite a lot of them. Many of them are restricted to exactly where we would expect them to be—within the lipid bilayer.” Binding of propofol and the model xenon yielded similar sets of residues, a finding that provides additional evidence for common molecular mechanisms of action of general anesthetics.
Drs. Joseph and Mincer will continue their research. “We are intrigued that so many different types of molecules actually create general anesthesia,” Dr. Mincer said. “The ability of the coarse-grained model, which by definition simplifies certain atomic-level interactions, to recapitulate and extend results from both theory and experiment is itself a proof of concept to some extent of minimal selectivity in the interactions of anesthetic ligands with ion channels. Trying to further delineate what exactly is important is something we want to explore further.”
The team envisions their work contributing alongside the efforts of researchers employing both theoretical and experimental methodologies to improve the understanding of how diverse agents produce general anesthesia. So stay tuned….