Ligand-Gated Ion Channel Gating Kinetics and the Opening/Closing Mechanism are Sensitive to Mutations Altering the Hydrophobicity of the Ion Conduction Pore

Abstract

The Cys-loop ligand-gated ion channel family includes the GABA-, glycine-, serotonin-, and nicotinergic acetylcholine receptors, which all play important roles in the nervous signal transduction pathway. They are critical targets for e.g. general anesthetics and alcohol, and all consist of five subunits organized around a central pore. Here, we investigate the prokaryotic Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) that is a homolog to the cys-loop superfamily. GLIC has the advantage of having several X-ray structures published, and we have previously used it for several different modeling studies. To better understand the relation between gating and the pore properties if ligand-gated channels in general, we have substituted several amino residues involved in the kinetics of open and closing of the channel, at different pH. We have characterized these substitutions using an in vitro electrophysiological functional assay (proton concentration response curves); expressing the substituted channels in Xenopus laevis oocytes for two-electrode voltage clamp experiments. We have also made single-channel recordings for investigating the kinetics (open, closing, and flickering behavior) of the substitutions, observed in silico. Our results indicate that the hydrophobicity of the pore is critical to determine both the balance and kinetics of the opening/closing process, and it is possible to alter the gating properties of the channel while still keeping it functional. This is an important step in the process of developing more effective anesthetics, with a better therapeutic window, but also to identify a possible molecular mechanism of how to reduce alcohol dependency.