Full mutational mapping of titratable residues helps to identify proton-sensors involved in the control of channel gating in the Gloeobacter violaceus pentameric ligand-gated ion channel

摘要

The Gloeobacter violaceus ligand-gated ion channel (GLIC) has been extensively studied by X-ray crystallography and other biophysical techniques. This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel (pLGIC) signal transduction. However, the GLIC is activated by lowering the pH and the location of its putative proton activation site(s) still remain(s) unknown. To this end, every Asp, Glu, and His residue was mutated individually or in combination and investigated by electrophysiology. In addition to the mutational analysis, key mutations were structurally resolved to address whether particular residues contribute to proton sensing, or alternatively to GLIC-gating, independently of the side chain protonation. The data show that multiple residues located below the orthosteric site, notably E26, D32, E35, and D122 in the lower part of the extracellular domain (ECD), along with E222, H235, E243, and H277 in the transmembrane domain (TMD), alter GLIC activation. D122 and H235 were found to also alter GLIC expression. E35 is identified as a key proton-sensing residue, whereby neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically to the orthosteric site, at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD. Author summary Pentameric ligand-gated ion channels are an important class of receptors that are involved in many neurological diseases. They have been extensively studied but a full understanding of their mechanism of action has yet to be achieved. In an effort to bypass obstacles in the research of human receptors, bacterial versions have been used to characterize the family’s structure-function relationship. One key bacterial receptor, known as GLIC, has lead the way in structural resolution of various mechanistic states along the gating pathway, yet its activation by protons is significantly less understood than its human counterparts. To define the site(s) involved in proton gating, we systematically mutated all titratable residues near the pH_(50)of activation: Asp, Glu, and His. We determined that a previously established His residue in the transmembrane domain is structurally important but likely plays little or no role in proton gating. We instead found that proton activation is a complex multiple loci mechanism, with the key contribution stemming from the coupling interface between the extracellular and transmembrane domain, with E35 acting as a key proton-sensing residue.