Altering the Open vs. Closed State Balance of the GLIC Ligand-Gated Ion Channel through Mutagenesis Enables Molecular Simulation of the Reversible Gating Process

Abstract

The prokaryotic Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) is an important template for studies of Cys-loop receptors in the human central nervous system. It is also a key model system to understand the transitions and stabilization of different conformations of membrane proteins through allosteric modulation - a ligand that opens the channel can either stabilize the open state, or destabilize the closed one. Capturing these processes on the molecular level will not only enable us to understand gating, but also make it possible to predict functional responses rather than merely binding properties. Over the last years, we have studied mutants of the GLIC channel that make it more similar to the human Cys-loop receptors both experimentally and in simulations, and shown how this provides evidence for a dual allosteric modulation mechanism with separate inhibitory and potentiating binding sites. Here, I will present our new results on mutations in the central GLIC pore. By modifying the hydrophobicity of one or several residues in the pore it is possible to alter the pore hydration level, which in turn has paramount effects on the conformation - molecular simulations show that it is possible to stabilize the channel either in the open or closed conformation independent of pH. By using ensemble molecular dynamics simulations covering several tens of microseconds, we show that it is possible to reversibly simulate transitions between conformations that are shown to actually conduct ions or block the current in simulations. In vivo electrophysiology and single-channel recordings on the mutants confirm this influence on the kinetics, and provide us with a way to couple molecular-level dynamics and kinetics to the experimentally observed equilibrium and transitions between conformations.