Computational and Biochemical Design of a Nanopore Cleavable by a Cancer‐Secreted Enzyme

Pittel, Ilya; Alper, Naomi; Yonai, Shiran; Basch, Shani; Blum, Leah; Bachur, Ayelet; Paas, Yoav

doi:10.1002/cbic.201402378

Cited by 1 publication

(1 citation statement)

References 73 publications

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“…The cDNA encoding the C. elegans GluClβWT subunit (GLC-2; see UniProt entry Q17328 for the ORF sequence) was prepared by reverse transcription of total C. elegans RNA and PCR amplification of the relevant ORF, which was subsequently cloned into a pcDNA3.1 vector. Single or double site-specific mutations were introduced as previously by using the QuikChange site-directed mutagenesis kit (Stratagene; Pittel et al, 2010, 2015). The entire ORF of all mutants was sequenced and subcloned into an original pcDNA3.1 vector.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%