Pentameric ligand-gated ion channels (pLGICs) play a primary role in synaptic transmission, and are modulated by a variety of endogenous molecules, including phospholipids, sterols, and fatty acids. pLGICs are also modulated by small molecule therapeutics (e.g. anesthetics and anti-epileptics). The structural mechanism by which phospholipids modulate pLGICs is poorly understood. Anionic phospholipids are allosteric modulators of mammalian pLGICs, and structural studies suggest that phospholipid binding sites overlaps with binding sites of small molecules such as neuroteroids. The goal of this project is to investigate the hypothesis that lipids and certain allosteric modulating drugs bind to specific sites on pLGICs, and that these drugs induce their modulatory effect through a positive, or negative, effect on lipid binding. To accomplish this goal, I will use a combination of cutting edge techniques, including native mass spectrometry (MS), covalent chemical modification, and patch- clamp recordings of giant liposomes of defined lipid composition. To apply these techniques, I will use the prototypical prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC), as a tractable model system. ELIC is an ideal system for MS and readily permits expression and purification of mutant proteins for biochemical and reconstitution studies. Work in the Cheng lab has measured direct binding of phospholipids to ELIC by MS, and demonstrated that specific binding of anionic phospholipids reduces desensitization in ELIC. Building upon this work, this research project will address two aims. The first is to determine the specificity and sites of phospholipid binding that mediate their modulatory effects on ELIC. I hypothesize that phospholipid head group charge determines the lipid binding affinity to ELIC, but that the structure of the hydrophobic tail (e.g. length and position of unsaturations) is the critical determinant of the native modulatory effect. Phospholipid binding affinity and stoichiometry will be determined by MS. The functionally-relevant binding sites for phospholipids will be determined using mutagenesis and chemical modification with methanethiosulfonate (MTS) reagents.
The second aim i s to elucidate the interaction between phospholipids and allosterically modulating drugs in relation to ELIC binding and modulation. Within this aim I will determine the sites of binding of allopregnanalone (alloP) in ELIC using photo-affinity labels, and then examine the effect this labeling (or non-covalent binding in MS) has on phospholipid binding. Preliminary results indicate that alloP enhances ELIC desensitization, and I hypothesize that alloP induces its pharmacologic effect by competing for binding of sites otherwise occupied by phospholipids. Functional studies in liposomes will determine whether alloP competitively or non-competitively antagonize anionic phospholipid effect. This work will be foundational in understanding the modulation of pLGICs by relevant small molecules. The experimental framework developed within this proposal will be critical in understanding the mechanism of channel modulation by other bioactive lipids and small molecule modulators.
There is a lack of understanding of how pharmaceutical modification and the native lipid environment of pentameric ligand-gated ion channels (pLGICs), a protein class essential in synaptic transmission, affects their function. I propose to elucidate the molecular mechanism of phospholipid and neuroteroid modulation of pLGICs. This work will provide an understanding of how native lipids and neuroteroids modulate pLGICs, which is instrumental in the design of novel small molecule modulators.
