Structural and Functional Studies Uncover Two Networks Stabilizing the Active Form of GLIC, a Bacterial Proton-Gated Pentameric Ion Channel

Abstract

The proton-activated pentameric ligand-gated ion channel from Gloeobacter violaceus GLIC can be used as a model system to study the structural and functional properties of its eukaryotic counterparts, which mediate signaling transduction in animal neuron cells by controlling permeation of ions flux gated by neurotransmitter binding. Here we used the Finite Difference Poisson-Boltzmann/Debye-Hückel method to predict the pKas of all the Asp and Glu in GLIC both in the active and resting states. Those residues with a high deviation of predicted pKas in both forms were titrated by Fourier-transform infra-red spectroscopy after reconstitution in lipid bilayers. The results showed that E35 is the main key proton-sensor residue. Examination of the active form structure shows that E35 interacts with T158 from Loop F. Here we verified that breaking this interaction hinders the proton-elicited currents. We next probed the interfacial crevice immediately below E35, shaped by Loop F, Q193 (pre-M1) and the neighboring M2-M3 loop, where a previously unnoticed hydrogen bond network may help maintaining the open channel. We show that breaking this hydrophilic network originating from Q193 favors the ion channel in a nonconductive conformation in the crystal state. Replacing side-chains of residues just below Q193, such as Y197 and I201, with other hydrophobic side-chains blocks the receptor in a locally closed form. Two signal transduction networks are proposed, both originating from the key proton-sensing residue E35, loop F and the pre-M1 region: i) one hydrophilic network extends to the M2-M3 loop from the neighboring subunit and ii) one hydrophobic network interacts with the Cys-loop and the M2-M3 loop of the same subunit. Due to the strong conservation of these loop regions, this model could be generalized to the entire pLGIC family.