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 alpha and beta subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClaR, 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 beta/alpha intersubunit interfaces, where the GluCl beta and GluCl alpha 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 alpha/alpha intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three a and two beta subunits arranged in a counter-clockwise beta-alpha-beta-alpha-alpha fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.