Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Abstract

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

A Functional Comparison of Homopentameric Nicotinic Acetylcholine Receptors (ACR-16) Receptors From Necator americanus and Ancylostoma ceylanicum

“…An important caveat is that this protein (accession code # AZS27833.1) differs from an earlier published sequence (accession code # ABW07339.1) by a single amino acid in the cys-loop (serine versus proline in ABW07339.1). This proline residue has been reported to be of critical importance for ligand-activation in the pLGIC family (Lummis et al, 2005;Rienzo et al, 2016), and highlights the role that a single residue can play in receptor activation.…”

Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity

“…Notably, the three M1-205 variants (Trp, Gly, and Pro) with expanded forms of the open-state intrasubunit cavity also exhibited decreased H + sensitivity, in accordance with a critical role for the extracellular-facing portion of M1 in gating and modulation ( 37 ). Previous studies in GLIC ( 38 , 39 ), nACh ( 40 , 41 ), and 5-HT 3 receptors ( 42 , 43 ) have shown that disruption of backbone N hydrogen bonding at Pro-204—the most conserved transmembrane residue among all pLGICs ( 44 )—is not only permissive, but necessary for channel gating. It is plausible that bulky or helix-disrupting residues at adjacent position 205 could account for an increased barrier to relieving the M1 kink, decreasing agonist sensitivity but increasing accessibility of M2 from the membrane.…”

Mutational analysis to explore long-range allosteric coupling and decoupling in a pentameric channel receptor

“…The conservation of the gating mechanism between bacterial and eukaryotic pLGICs is well documented by the available structures with the common allosteric regulatory sites for ligands and mutations (Sauguet et al, 2015; Bertozzi et al, 2016; Rienzo et al, 2016), together with the allosteric compatibility between eukaryotic and prokaryotic ECD/TMD domains to form functional chimeras (Duret et al, 2011; Moraga-Cid et al, 2015; Laverty et al, 2017). It is therefore tempting to speculate that the pre-activation transition of GLIC that we characterize here might have counterparts in human neurotransmitter-gated receptors.…”