Fine-tuning the pentameric ligand-gated ion channel response via the lipid bilayer

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My research program is focused on understanding the mechanisms by which lipids influence membrane protein function. Specifically, I am interested in the lipid sensitivities of a super-family of proteins called pentameric ligand-gated ion channels (pLGICs). These ion channels are typically found in post-synaptic membranes, where they convert a chemical signal (the neurotransmitter) into an electrical response (change in membrane potential). One fascinating feature of pLGICs is that channel gating occurs on the millisecond time scale with even subtle changes in gating having profound biological effects. In addition, the activities of these ion channels are highly sensitive to lipids suggesting that lipids may play a role in pLGIC function. My research program has undergone a major shift due to recent advances in cryo-electron microscopy, which now allow us to solve high resolution structures of membrane proteins embedded in lipid bilayers (nanodiscs). During my recent sabbatical, I developed methods for reconstituting functional nicotinic acetylcholine receptors (nAChRs) into lipid nanodiscs and solved structures at high enough resolution to identify 11 lipid binding sites. Through collaborations, I also recently solved a relatively high-resolution x-ray crystal structure of the prokaryotic pLGIC, ELIC and identified a lipid binding site that plays a role in ELIC function. This binding site was functionally characterized leading to a relatively detailed model of how lipid binding influences ELIC desensitization. The work described in this research grant proposal will build on these exciting seminal findings. The goal of this proposal is to elucidate and compare the mechanisms by which lipids alter the function of three pLGICs, the nAChR, GLIC and ELIC. Specifically, we will we focus on the mechanisms by which lipid binding to all three pLGICs alters function. We will 1) define the endogenous lipids that exist in the microenvironments surrounding each pLGIC, 2) characterize how these and other lipids influence channel gating, 3) explore the binding of these and other lipids to each pLGIC using molecular dynamics (MD) simulations, 4) solve pLGIC structures in different membranes to explore both lipid binding specificity and the effects of lipid binding on structure, and 5) use a combination of mutagenesis and electrophysiology to validate mechanistic hypothesis. We can now for the first time solve structures of membrane proteins in lipid environments. We can visualize lipid binding sites and then explore the functional consequences of lipid binding using sophisticated biophysical techniques. My lab is poised to make major breakthroughs in our understanding of lipid-protein interactions. The proposed studies will not only lead to major breakthroughs in our understanding of lipid-pLGIC interactions but will also provide an exceptional environment for the training of post-graduate, graduate, and undergraduate trainees.