Probing Allosteric Networks in Ligand-Gated Ion Channel Gating by Electrophysiology and Molecular Dynamics in the Model Receptor GLIC

In all kingdoms of life, pentameric ligand-gated ion channels mediate electrical activity in response to chemical stimuli including pH, neurotransmitters, alcohols, and a wide range pharmacological agents. Recent biochemical structural studies this family receptors have identified putative binding sites for various agonists modulators, but leave open questions as the mechanisms coupling between ligand pore opening. Here, we used voltage-clamp electrophysiology molecular dynamics investigate transmembrane gating determinants GLIC, structurally accessible model channel from cyanobacteria. As measured by two-electrode electrophysiology, mutations or at subunit interface enhanced proton sensitivity, had opposing effects on ethanol potentiation. Molecular simulations indicated these different mechanisms, primarily involving either extracellular blooming electrostatic networks. Ethanol flooding closed states substantiated presence multiple modulatory sites, with modifying state-dependent occupancies regions. Modifications both closed- open-state models were required stable extended simulations, highlighting tractable uncertainties experimental structures. Finally, comparative modeling eukaryotic members provided rationale differential sensitivities allosteric modulators. These results reveal distinct networks influencing modulation family, processes likely critical elucidating evolution designing novel therapeutics.