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

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

Trapping of ivermectin by a pentameric ligand-gated ion channel upon open-to-closed isomerization.

Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C. elegans heteromeric GluClα/ß receptor, it cannot activate a homomeric receptor composed of the C. elegans GluClß subunits. To understand this incapability, we generated a homopentameric α7-GluClß chimeric receptor that consists of an extracellular ligand-binding domain of an α7 nicotinic acetylcholine receptor known to be potentiated by IVM, and a chloride-selective channel domain assembled from GluClß subunits. Application of IVM prior to acetylcholine inhibited the responses of the chimeric α7-GluClßR. Adding IVM to activated α7-GluClßRs, considerably accelerated the decline of ACh-elicited currents and stabilized the receptors in a non-conducting state. Determination of IVM association and dissociation rate constants and recovery experiments suggest that, following initial IVM binding to open α7-GluClßRs, the drug induces a conformational change and locks the ion channel in a closed state for a long duration. We further found that IVM also inhibits the activation by glutamate of a homomeric receptor assembled from the C. elegans full-length GluClß subunits.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel.

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and ß subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at ß/α intersubunit interfaces, where the GluClß and GluClα subunits, respectively, contribute the "principal" and "complementary" components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits' cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two ß subunits arranged in a counterclockwise ß-α-ß-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.