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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2017
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382172/ https://www.ncbi.nlm.nih.gov/pubmed/28428744 http://dx.doi.org/10.3389/fnmol.2017.00092 |
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author | Degani-Katzav, Nurit Gortler, Revital Weissman, Marina Paas, Yoav |
author_facet | Degani-Katzav, Nurit Gortler, Revital Weissman, Marina Paas, Yoav |
author_sort | Degani-Katzav, Nurit |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5382172 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-53821722017-04-20 Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin Degani-Katzav, Nurit Gortler, Revital Weissman, Marina Paas, Yoav Front Mol Neurosci Neuroscience 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. Frontiers Media S.A. 2017-04-06 /pmc/articles/PMC5382172/ /pubmed/28428744 http://dx.doi.org/10.3389/fnmol.2017.00092 Text en Copyright © 2017 Degani-Katzav, Gortler, Weissman and Paas. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Neuroscience Degani-Katzav, Nurit Gortler, Revital Weissman, Marina Paas, Yoav Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin |
title | Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin |
title_full | Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin |
title_fullStr | Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin |
title_full_unstemmed | Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin |
title_short | Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin |
title_sort | mutational analysis at intersubunit interfaces of an anionic glutamate receptor reveals a key interaction important for channel gating by ivermectin |
topic | Neuroscience |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382172/ https://www.ncbi.nlm.nih.gov/pubmed/28428744 http://dx.doi.org/10.3389/fnmol.2017.00092 |
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