Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

摘要: The broad-spectrum anthelmintic drug ivermectin activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing continuous inflow chloride ions sustained membrane hyperpolarization. These effects suppress nervous impulses vital physiological processes in parasitic nematodes. GluClRs are pentamers. Homopentameric assembled from the Caenorhabditis elegans GluClα (GLC-1) subunit can inherently respond to but not (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies both ligands, independently each other. Glutamate bind at interface between adjacent subunits, far away other; extracellular ligand-binding domain, ion-channel pore periphery. To understand importance putative intersubunit contacts located outside binding sites, we introduced mutations interfaces, these two binding-site types. Then, determined effect on activation mutant by ivermectin. Amongst mutations, characterized α-subunit point mutation close ivermectin-binding pocket, end first transmembrane helix (M1). This (αF276A) moderately reduced sensitivity GluClαF276A/βWT receptor glutamate, slightly decreased subunits’ cooperativity response glutamate. αF276A drastically significantly increased We suggest that this reduces efficacy channel gating, impairs integrity likely disrupting important interactions tip M1 M2‑M3 loop subunit. hypothesize physical contact M2-M3 tunes relative orientation helices M1, M2 M3 optimize opening. Interestingly, pre-exposure GluClαF276A/αWT subthreshold concentration recovered infer retained its positive modulation activity constraining a preopen sensitive with no need for aforementioned disrupted loop.