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

Degani-Katzav, Nurit; Gortler, Revital; Gorodetzki, Lilach; Paas, Yoav

doi:10.1073/pnas.1423753113

Cited by 22 publications

(31 citation statements)

References 76 publications

(119 reference statements)

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Section: Resultsmentioning

confidence: 99%

“…We have recently shown that CHO cells transfected with the wild type GluClα subunit (αWT) alone display very weak responses to 10 mM Glu but robust responses to 500 nM IVM (Degani-Katzav et al, 2016). It was also shown that CHO cells transfected with the wild type GluClβ subunit (βWT) alone display very weak, rare responses to 10 mM Glu (Degani-Katzav et al, 2016). Others also reported irresponsiveness to Glu in human embryonic kidney (HEK) cells transfected with the wild type GluClβ subunit alone (Slimko et al, 2002; Frazier et al, 2013; Daeffler et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

“…Most recently, we succeeded to obtain responses of a few hundred picooamperes in CHO cells transfected with the WT GluClβ subunit alone using the X-tremeGENE HP DNA Transfection Reagent (Roche Life Science); but, in this case, we challenged the cells with 100 mM Glu, and only 4 of 46 cells responded (Degani-Katzav et al, 2017). In contrast, CHO cells co-transfected with both WT GluCl α and β subunits commonly display robust responses to both Glu and IVM (Degani-Katzav et al, 2016). These results are summarized in Table 2.…”

Section: Resultsmentioning

confidence: 99%

“…Yet, Glu elicits current responses in homomeric C. elegans GluClαRs when applied after activation by IVM, indicating that IVM binding to the homomeric C. elegans GluClαR induces a conformational change that couples Glu binding at GluCl α/α intersubunit interfaces to the ion-channel gate (Etter et al, 1996). Recently, it was demonstrated that a C. elegans heteromeric GluClα/β receptor, whose β subunits were engineered to carry the α-subunit’s Cys and β8β9 loops (Figure 1A), readily responds to Glu, with no need of IVM pre-association (Degani-Katzav et al, 2016). Since the GluCl α and β subunits share the same β1β2-loop sequence, all the coupling loops originating from the ligand-binding domain of this heteromeric mutant receptor (β1β2, Cys and β8β9 loops in each subunit) have the sequence of the α subunit.…”

Section: Introductionmentioning

confidence: 99%

“…Since the GluCl α and β subunits share the same β1β2-loop sequence, all the coupling loops originating from the ligand-binding domain of this heteromeric mutant receptor (β1β2, Cys and β8β9 loops in each subunit) have the sequence of the α subunit. Hence, it was concluded that these α-subunit’s coupling loops are inherently capable of transducing motions in the Glu-binding site to gating motions in the ion-channel pore (Degani-Katzav et al, 2016). Moreover, a mutation inside the IVM binding pocket (αL279W) increased the sensitivity of the C. elegans GluClα/βR to both IVM and Glu (Degani-Katzav et al, 2016), suggesting that the IVM and Glu binding sites in the C. elegans GluClα/β heteromeric receptor are allosterically coupled.…”

Section: Introductionmentioning

confidence: 99%

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

Degani-Katzav

Gortler

Weissman

et al. 2017

Front. Mol. Neurosci.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Degani-Katzav

Gortler

Weissman

et al. 2017

Front. Mol. Neurosci.

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Practical Considerations for the Use of DREADD and Other Chemogenetic Receptors to Regulate Neuronal Activity in the Mammalian Brain

Aldrin-Kirk

Björklund

2019

Methods in Molecular Biology

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Effect of sublethal concentrations of the antiparasitic ivermectin on the polychaeta species Hediste diversicolor: biochemical and behavioral responses

2021

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Pharmaceutical drugs have emerged as major micropollutants in aquatic ecosystems. Their presence has been systematically reported in monitoring survey, and their wide distribution and constant presence in the wild is a direct consequence of their massive use, in both human and veterinary therapeutics. Drugs used to treat parasitic infections in livestock are major contaminants, given the amounts in which they are administered, and reach the aquatic compartment in high amounts, where they may affect non target species. Some of these drugs are prone to nd their nal deposit in sediments of estuarine areas, exerting their toxic effects preferentially at these locations. Sediment dwelling organisms of coastal areas, such as polychaetes, are especially prone to have their major physiological functions compromised. Ivermectin is one of the most used antiparasitic drugs, and its effects are not limited to biochemical traits, but also behavioral features may be compromised considering their neurotoxic actions. Despite these putative effects, little is known about their toxicity on polychaetes. The present study aimed to characterizes the toxicity of realistic levels of ivermectin on the polychaete Nereis diversicolor, in biochemical and behavioral terms. The obtained results showed that low levels of ivermectin are capable of causing signi cant disturbances in mobility and burrowing activity of exposed worms, as well as alterations of metabolic and anti-oxidant defense e cacy of exposed animals, suggesting that its environment presence may mean a major environmental concern.

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

Cited by 22 publications

References 76 publications

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

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

Practical Considerations for the Use of DREADD and Other Chemogenetic Receptors to Regulate Neuronal Activity in the Mammalian Brain

Effect of sublethal concentrations of the antiparasitic ivermectin on the polychaeta species Hediste diversicolor: biochemical and behavioral responses

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