The pentameric ligand gated ion channel super family plays a central role in fast synaptic communication between neurons and at the neuromuscular junction. Extensive studies on the prototypic pLGIC, the Torpedo nicotinic acetylcholine receptor (nAChR) have revealed an exquisite lipid sensitivity, with the nAChR adopting a novel uncoupled conformation in membranes lacking activating anionic and neutral lipids. The lipid-exposed transmembrane alpha-helix, M4, in each homologous subunit likely acts as a lipid sensor. One model proposes that activating lipids promote M4 “binding” to the adjacent alpha-helices, M1 and M3, to enhance interactions between the M4 C-terminus and the Cys-loop of the agonist-binding domain, with such interactions promoting coupling between the agonist site and channel gate. The first part of my thesis indirectly tests this hypothesis by exploring the effects of membrane hydrophobic thickness on nAChR function. Specifically, I tested the hypothesis that thicker membranes, which should promote alignment of M4 parallel to M1/M3 and thus helix-helix interactions, favor a coupled conformation. Although I found that the nAChR is uncoupled in all membranes tested, regardless of hydrophobic thickness, thicker membranes promote transitions from uncoupled to ultimately the desensitized state over the minutes to hours time frame. In contrast to anionic lipids, which influence function primarily via a conformational selection mechanism, membrane hydrophobic thickness influences function via a kinetic mechanism - thick membranes lower the activation energy between uncoupled and coupled conformations to promote conformational transitions. In the second part of my thesis, I used the two prokaryotic homologs, GLIC and ELIC, to explore how amino acid interactions at the interface between M4 and M1/M3 influence channel activity. Alanine scanning mutagenesis of this interface shows that disruption of almost any interaction in GLIC leads to a loss of folding and/or function, while analogous mutations in ELIC typically lead to no change or produce gains in function. Sequence comparisons with other members of the pLGIC superfamily suggest that the transmembrane domains of GLIC and ELIC represent two distinct archetypes. Each archetype may strike a different balance between the need for strong M4 binding to M1/M3 to promote folding and pentamer assembly, and the need for weaker interactions that allow for greater conformational flexibility during function.

In lipid membranes lacking activating lipids, the nicotinic acetylcholine receptor adopts an uncoupled conformation that binds ligand, but does not transition into an open conformation. Understanding the mechanisms of lipid-dependent uncoupling is essential to understanding lipid-nAChR interactions, which may be implicated in pathological conditions such as nicotine addition. Here, I tested two structural features of a proposed uncoupling method to elucidate the mechanism of lipid-dependent uncoupling. First, infrared measurements and electrophysiological characterization performed in prokaryotic homologues indicate that lipid sensitivity is largely controlled by the most peripheral α-helix in the transmembrane domain, M4. My data show that tighter association of M4 with the adjacent M1 and M3 transmembrane α-helices decreases a receptor’s propensity to adopt a lipid-dependent uncoupled conformation. Second, I indirectly tested the hypothesis that uncoupling results from a conformational change at the extracellular/transmembrane domain interface that leads to an increased separation between the two domains and ultimately to a constriction of the channel pore. Finally, biophysical studies presented in this dissertation shed light on the complex binding of a number of non-competitive channel blockers to the nicotinic acetylcholine receptor channel pore in both the resting and desensitized states. The data provide further insight into the structural rearrangements that occur upon uncoupling of ligand binding and gating in the nicotinic acetylcholine receptor.

This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel. In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.