Abstract
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 β 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 β/α intersubunit interfaces, where the GluClβ 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 β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.
Highlights
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 aforementioned crystallographic observations [23] are consistent with earlier studies showing that Glu elicits current responses in homomeric GluClαRs only when applied after activation by IVM [14], which gives rise to the following question: Could an α/α intersubunit interface be formed in a heteromeric assembly, bind Glu, and functionally participate in the activation process even without IVM preassociation? To resolve this question, we clarified here the stoichiometry and positions of the α and β subunits in GluClα/βR heteromeric assemblies that carry mutations in both the putative Glu- and IVM-binding pockets
glutamategated chloride-selective receptor (GluClR) are unique to invertebrates, yet they are pharmacologically important because they serve as targets for ivermectin, an anthelmintic drug used to treat humans suffering from filarial diseases
Summary
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. A recent 3D crystal structure of a truncated homomeric GluClαR (GluClαcrystR; Protein Data Bank ID code 3RIF) shows that when IVM is bound at the five α/α intersubunit interfaces in the ion-channel pore periphery, Glu is lodged at the five α/α intersubunit interfaces in the ligand-binding domain (LigBD) [23] (Fig. 1A) These Glu-binding sites are homologous to the neurotransmitter/agonist-binding sites of other Cys-loop receptors [1, 2, 24], bacterial homologs of Cys-loop receptors [25,26,27,28,29,30], and ACh-binding proteins [31,32,33,34]. The aforementioned crystallographic observations [23] are consistent with earlier studies showing that Glu elicits current responses in homomeric GluClαRs only when applied after activation by IVM [14], which gives rise to the following question: Could an α/α intersubunit interface be formed in a heteromeric assembly, bind Glu, and functionally participate in the activation process even without IVM preassociation? To resolve this question, we clarified here the stoichiometry and positions of the α and β subunits in GluClα/βR heteromeric assemblies that carry mutations in both the putative Glu- and IVM-binding pockets
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