Functional prokaryotic–eukaryotic chimera from the pentameric ligand-gated ion channel family

Abstract

Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.Gloeobacter violaceus | allosteric effector | evolution | fusion protein | membrane protein P entameric ligand-gated ion channels (pLGICs) mediate chemo-electric signal transduction in animals, thereby fulfilling key physiological functions, including neurotransmission. They are composed of five identical or homologous subunits and carry one to five agonist binding sites on their extracellular domain (ECD) that govern the opening/closing motion of the ion channel within the transmembrane domain (TMD, composed of four helices labeled M1-M4). In human, ≈40 genes code for pLGIC subunits that are organized into two phylogenetic subclasses: cationic excitatory channels, such as nicotinic acetylcholine (nAChR) and 5HT 3 receptors, and anionic inhibitory channels, such as glycine and GABA A receptors (1). More recently, several new members of the family have been discovered in prokaryotes (2), such as the homologous protein from Gloeobacter violaceus (GLIC), which forms a homopentamer functioning as a proton-gated ion channel (3).Eukaryotic and prokaryotic receptors clearly display homologous structures, characterized by a highly conserved β-sandwichfolded ECD coupled to an all-helix TMD, as described by electron microscopy observation of the Torpedo nAChR (TnAChR) (4), and the X-ray structures of the acetylcholine binding protein (...

Allosteric and hyperekplexic mutant phenotypes investigated on an α 1 glycine receptor transmembrane structure

“…They do not impair the allosteric coupling, but certainly modulate the functional properties. For instance, we showed that performing the F119Y and M121F mutations on Lily results in an acceleration of the desensitization kinetics (16). Extensive mutagenesis work has shown that the entire interface is involved in fine tuning of the allosteric response to agonist (18,19).…”

“…Coordinates and structure factors were deposited in the Protein Data Bank (PDB) with PDB ID 4X5T. Whole-cell and single-channel patch-clamp electrophysiology was performed as previously described (16). Details are provided in SI Materials and Methods.…”

“…Indeed, GLIC crystal packings involve mainly interactions between the ECD and the short cytoplasmic M3-M4 loop, but only limited interaction with the TMD, largely shielded from the solvent by the detergent. In previous work, we designed a chimera composed of the ECD of GLIC fused to the TMD of the human α 1 GlyR, where the ICD of the α 1 GlyR was replaced by the short linker (SQP motif) found in GLIC (16). In this chimera, mutations Y119F and F121M were introduced (loop 7) and the C-terminal tail of the α 1 GlyR was substituted for that of GLIC to increase the structural complementarities between the ECD and the TMD.…”

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

“…2 (see also Table 1). Note that the proton binding sites for GLIC activation are located primarily in the ECD (47), although an intramembranous His residue on M2 is essential (45,48). Given the long distance between the sites of mutation and the agonist (proton) binding sites, the shifts in ligand sensitivity likely reflect changes in the ability to couple agonist binding to channel gating.…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…Furthermore, their remarkable sensitivity to alcohols, general anesthetics, and other clinically relevant compounds (11,12) make them attractive targets for drug design. Remarkably, chimeras of GLIC with other eukaryotic members of the LGIC family retain the functional properties of the individual domains, strongly suggesting that the pathway for allosteric communication is essentially conserved across prokaryotic and eukaryotic channels (13,14).…”