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

Abstract

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/β (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/βWT 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/βWT 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.

Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus

“…contortus , such as TM3-G36’ [30] and TM1-P230 [31], and in the α subunit of C . elegans , such as TM1-L279 and TM1-F276 [32, 33]. The glutamate binding site and TM3 domain are also sites that harbour mutations in wild ML-resistant strains of C .…”

Residues in the 1st Transmembrane-Spanning Helix Are Important for GABAAρ Receptor Function

“…This hypothesis is consistent with the results from the non-Ala substitutions which revealed aromaticity was needed for the residue at position 284. Aromaticity here is conserved across equivalent residues in other GABA A R subunits and in most pLGICs ( Figure 3 ), and mutation to Ala of this residue in GluCl (F276A) and GlyR (Y233A) yields non-functional receptors, while substitution to another aromatic does not [ 20 , 21 ]. Thus, these π–π and/or CH–π interactions with the Cys-loop Pro are likely conserved and critical for receptor function in the whole pLGIC family.…”

Получение Авермектинов: Биотехнологии И Органический Синтез

“…Однако авермектины безопасны для млекопитающих, так как не могут преодолеть гематоэнцефалический барьер и достичь ГАМК А -чув-ствительные Cl-каналы в центральной нервной системе (105). Как известно, GluCl-каналы и GABA A -рецепторы входят в семейство Cys-петлевых рецепторов, включающее также глициновые, никотиновые и серотониновые (5-HT3) ионотропные рецепторы (101,106,107), с которыми авермектины и мильбемицины также взаимодействуют, проявляя, однако, меньшую аффинность. Обнаружено также взаимодействие ивермектина с рецептором P2X4 (108).…”