Unravelling the Conformational Plasticity of the Extracellular Domain of a Prokaryotic nAChR Homologue in Solution by NMR

Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution

Protein expression of nonfunctional mutants.

<p>Rabbit anti-HA tag Alexa Fluor-695 immunostaining results of the mutants that produced no detectable current in functional tests, compared to Wt and GFP-alone injected oocytes. Left, colored merge of GFP and Alexa-695 and right, the Alexa-695 imaging alone. Both D122N and H235A show no expression. GLIC, <i>G</i>. <i>violaceus</i> ligand-gated ion channel; GFP, green fluorescent protein; HA, human influenza hemagglutinin; Wt, wild-type.</p

Identification of Cinnamic Acid Derivatives As Novel Antagonists of the Prokaryotic Proton-Gated Ion Channel GLIC

Pentameric ligand gated ion channels (pLGICs) mediate signal transduction. The binding of an extracellular ligand is coupled to the transmembrane channel opening. So far, all known agonists bind at the interface between subunits in a topologically conserved “orthosteric site” whose amino acid composition defines the pharmacological specificity of pLGIC subtypes. A striking exception is the bacterial proton-activated GLIC protein, exhibiting an uncommon orthosteric binding site in terms of sequence and local architecture. Among a library of Gloeobacter violaceus metabolites, we identified a series of cinnamic acid derivatives, which antagonize the GLIC proton-elicited response. Structure–activity analysis shows a key contribution of the carboxylate moiety to GLIC inhibition. Molecular docking coupled to site-directed mutagenesis support that the binding pocket is located below the classical orthosteric site. These antagonists provide new tools to modulate conformation of GLIC, currently used as a prototypic pLGIC, and opens new avenues to study the signal transduction mechanism