Transmembrane Structural Determinants of Alcohol Binding and Modulation in A Model Ligand-Gated Ion Channel

Pentameric ligand-gated ion channels are heavily implicated in neurological effects of alcohol, yet a structural understanding of this process remains limited by a lack of high-resolution data for pharmacologically relevant receptors. The prokaryotic ligand-gated ion channel GLIC is a potentially valuable model system whose structure has been determined in multiple conformations and bound to various ligands. In particular, modification of a key transmembrane position in GLIC rendered it potently sensitive to potentiation, and enabled co-crystallization with ethanol as well as other anesthetic agents. To elucidate alcohol interactions with channel structure and function, we substituted a variety of amino acids in the ethanol site and channel pore, expressed the mutated channels in frog oocytes, and measured their gating and modulation properties by two-electrode voltage-clamp electrophysiology. We compared the resulting changes in agonist and modulator sensitivity with standard amino acid properties and molecular modeling of the predicted binding site and gating transitions. We identified structural determinants of binding that may reflect distinct properties of alcohols relative to classic drugs, and could provide insight into other low-affinity modulators such as anesthetics and lipids. We further found that modifying the ethanol pocket removed or even reversed effects of mutations in the ion channel pore, implicating tight coupling between the allosteric and active sites. Our ongoing work aims to integrate functional data with computational models of receptor binding and channel gating.