Direct Lipid Binding in a Pentameric Ligand-Gated Ion Channel Assessed by Native Mass Spectrometry

Abstract

Pentameric ligand-gated ion channels (pLGICs) such as the nicotinic acetylcholine receptor or the prokaryotic homologue, Erwinia ligand-gated ion channel (ELIC), require anionic phospholipids for agonist-induced channel activation. Whether this requirement is mediated by direct lipid binding events to specific sites is not known, and understanding this phenomenon is essential for the development of lipophilic allosteric modulators of pLGICs. We used native mass spectrometry (MS) coupled with thermal stability and liposome flux assays to examine direct binding of phospholipids to ELIC and the functional effects of binding. ELIC from E. coli membranes co-purify with bound phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) lipids, showing selectivity for PG lipids. Native MS analysis of de-lipidated ELIC using exogenous phospholipids reveal that anionic phospholipids such as PG directly bind to ELIC with higher affinity than PE or phosphatidylcholine (PC). By correlating the stoichiometry of binding with lipid concentration, we determine that two bound PG lipids per subunit are required to stabilize ELIC from thermal denaturation. To identify the sites of PG binding, single and double mutations of five arginine residues at the transmembrane domain interfacial region were generated. Analysis of these mutants reveal two clusters of positively charged residues at both extracellular and intracellular leaflets that are essential for PG binding and thermal stabilization. Ongoing single particle cryo-EM studies of ELIC reconstituted in nanodiscs of defined lipid composition is anticipated to corroborate these sites of lipid binding, and may reveal the structure of uncoupled and activated/desensitized ELIC channels induced by these lipid-binding events.