Project Summary/Abstract Dr. Mark Arcario is a cardiothoracic anesthesiologist whose long-term goal is to be an independent physician- scientist studying molecular mechanisms of allostery in ion channels. His research background is in computational biophysics of membrane proteins with a focus on the effects of lipids and lipophiles on membrane protein structure and function. He has a special interest in pentameric ligand-gated ion channels (pLGICs) and mechanisms of anesthetic action. Several endogenous lipids, including neurosteroids, phospholipids, and fatty acids, are strong allosteric modulators of pLGICs and it has been suggested that drugs, including anesthetics, exploit these innate regulatory mechanisms. It is not well-known how lipophiles, which bind at the periphery of the protein, alter channel function and equilibrium between functional states. Such a molecular mechanism is essential for the rational, structure-based design of new drugs targeting this family of channels. To address this knowledge gap, the candidate will use a combination of innovative molecular dynamics (MD) techniques to characterize state-dependent structural ensembles of prokaryotic pLGIC, ELIC (Erwinia ligand-gated ion channel), and the human 7 nicotinic acetylcholine receptor (nAChR). Aim 1 will determine how the conformation of the lipid-facing, fourth transmembrane (M4) helix varies with the functional state of the channel (i.e., resting, activated, desensitized), using computational electrophysiology methods. Aim 2 will characterize the binding site of docosahexaenoic acid (DHA), a polyunsaturated fatty acid, and how binding of DHA causes inhibition using multiscale simulation and free energy calculations. This work will yield novel insight into state-dependent dynamics of pLGICs as well as establish a molecular model of lipophilic allosteric modulation. This research will be conducted under Dr. Wayland Cheng, an expert in pLGIC structure and function with a focus on lipid binding and modulation, along with co-mentor, Dr. Grace Brannigan, an expert in computational biophysics with a focus on MD method development and application to membrane proteins. An advisory committee comprised of Dr. Alex Evers, an expert in anesthetic pharmacology, Dr. Baron Chanda, an expert in ion channel thermodynamics and gating, and Dr. Jerome Hénin, an expert in free energy calculations, will aid in the scientific development of the candidate. The research plan will allow the candidate to develop skills in free energy calculations and computational electrophysiology, which will prepare him for a career in computational biophysics of ion channels. In addition, involvement in career development activities during this award will prepare the candidate for independence by the end of the award. The Department of Anesthesiology at Washington University in St. Louis, a leader in academic anesthesiology, provides an excellent and supportive environment for ion channel research and development of physician-scientists.

Project Narrative Responsible for fast synaptic transmission throughout the nervous system, pentameric ligand-gated ion channels (pLGICs) are the targets of many drugs essential to the modern practice of anesthesiology and critical care medicine, but drug development in this field has been stagnant for many years. In this proposal, I will use advanced computational techniques to understand the structural determinants of pLGIC modulation via lipids, which have been suggested to have molecular mechanisms that overlap with anesthetics. The outcome of this work will be a detailed molecular model of how lipophilic species allosterically modulate pLGICs, opening new avenues for drug discovery in anesthesia.