Electrostatics, proton sensor, and networks governing the gating transition in GLIC, a proton-gated pentameric ion channel

Abstract

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure–function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson–Boltzmann/Debye–Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pKa close to the experimental pH50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pKa at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain–transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC’s gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

Simulating ion channel activation mechanisms using swarms of trajectories

“…Functionally, the GLIC channel is known to activate due to a change in pH; fully converting from open to closed within the range 4.6–7, with pH 50 ~5.3 . Figure b indicates which residues in the ECD have been predicted to change protonation state, identified through pK a calculations and X‐ray crystallographic data for ion binding sites, confirmed by mutagenesis and electrophysiology studies, as well as more recently via Fourier Transform Infrared Resonance (FTIR) spectroscopy . Experiments have also yielded other mechanistic information, including X‐ray structures of a possible intermediate state, structural changes with electron paramagnetic resonance (EPR) spectroscopy, and kinetic analysis identifying residues contributing during conformational transitions in the pLGIC family .…”

Characterization of the dynamic resting state of a pentameric ligand-gated ion channel by cryo-electron microscopy and simulations

“…Similarly, a counter-clockwise twist of the ECD with respect to the TMD has been proposed as an initiating step in pentameric ion channel activation [37] , [38] , [39] . In parallel to the ECD radius, domain twist fluctuated over a relatively wide range (from -13° to -28°) in simulations of the pH 7 cryo-EM structure (Fig.…”

“…The most prominent geometric change in pentameric channel gating that enables ion flux is the expansion of the transmembrane pore, with the upper region of each M2 helix and the M2-M3 loop spreading outward from the conduction pathway [37] .…”

Structural and molecular insight into the pH-induced low-permeability of the voltage-gated potassium channel Kv1.2 through dewetting of the water cavity

“…Electrical signals in neurons are generated by sequential gating of several voltage-gated ion channels on their cell membranes. The opening and closing of these channels are not only sensitively controlled by membrane potentials in general, but also respond to the intra-and extracellular conditions, such as chemicals [1,2], mechanical pressure [3], temperature [4], and proton concentrations [5]. Among these channels, the voltage-gated potassium channels (Kv) are selectively permeable to potassium ions and repolarize the membrane potential in response to depolarizing voltage [6].…”