scholarly journals Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions

2020 ◽  
Vol 117 (28) ◽  
pp. 16283-16291 ◽  
Author(s):  
Makoto Ihara ◽  
Shogo Furutani ◽  
Sho Shigetou ◽  
Shota Shimada ◽  
Kunihiro Niki ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Receptors ◽  
Bombus Terrestris ◽  
Transmembrane Protein ◽  
Molecular Targets ◽  
Fruit Fly ◽  
Functional Expression ◽  
Insect Pollinators

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression inXenopus laevisoocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

scholarly journals Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

2004 ◽  
Vol 123 (6) ◽  
pp. 697-708 ◽  
Author(s):  
Paul J. Groot-Kormelink ◽  
Steven D. Broadbent ◽  
James P. Boorman ◽  
Lucia G. Sivilotti
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Receptors ◽  
Functional Characterization ◽  
Functional Expression ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Neuronal Nicotinic Receptors ◽  
Channel Mutation ◽  
Tandem Constructs

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.

Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells

2014 ◽  
Vol 1542 ◽  
pp. 1-11 ◽  
Author(s):  
Andreas H. Rasmussen ◽  
Dorte Strøbæk ◽  
Tino Dyhring ◽  
Marianne L. Jensen ◽  
Dan Peters ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hek293 Cells ◽  
Pharmacological Characterization ◽  
Nicotinic Acetylcholine

scholarly journals Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy

2020 ◽  
Vol 47 (6) ◽  
pp. 800-809 ◽  
Author(s):  
Maxime Cadieux-Dion ◽  
Simone Meneghini ◽  
Chiara Villa ◽  
Dènahin Hinnoutondji Toffa ◽  
Ronny Wickstrom ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Missense Variant ◽  
Functional Expression ◽  
Functional Studies ◽  
Sorting Intolerant From Tolerant ◽  
First Case ◽  
And Function

Abstract:Purpose:Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy.Method:We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene.Results:We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4β2 (wild-type), α4β2-Thr26Met (homozygote), and α4β2/β2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation.Conclusions:This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

Molecular characterization of the specificity of interactions of various neurotoxins on two distinct nicotinic acetylcholine receptors

2000 ◽  
Vol 393 (1-3) ◽  
pp. 197-204 ◽  
Author(s):  
Denis Servent ◽  
Stéphanie Antil-Delbeke ◽  
Carole Gaillard ◽  
Pierre-Jean Corringer ◽  
Jean Pierre Changeux ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine

scholarly journals Classifying neuronal subclasses of the cerebellum through constellation pharmacology

2016 ◽  
Vol 115 (2) ◽  
pp. 1031-1042 ◽  
Author(s):  
Kigen J. Curtice ◽  
Lee S. Leavitt ◽  
Kevin Chase ◽  
Shrinivasan Raghuraman ◽  
Martin P. Horvath ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Gaba Receptors ◽  
Acetylcholine Receptors ◽  
Granule Cells ◽  
Functional Expression ◽  
Muscarinic Acetylcholine Receptors ◽  
Receptor Subtypes ◽  
Mouse Cerebellum ◽  
The Brain

A pressing need in neurobiology is the comprehensive identification and characterization of neuronal subclasses within the mammalian nervous system. To this end, we used constellation pharmacology as a method to interrogate the neuronal and glial subclasses of the mouse cerebellum individually and simultaneously. We then evaluated the data obtained from constellation-pharmacology experiments by cluster analysis to classify cells into neuronal and glial subclasses, based on their functional expression of glutamate, acetylcholine, and GABA receptors, among other ion channels. Conantokin peptides were used to identify N-methyl-d-aspartate (NMDA) receptor subtypes, which revealed that neurons of the young mouse cerebellum expressed NR2A and NR2B NMDA receptor subunits. Additional pharmacological tools disclosed differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazloepropionic, nicotinic acetylcholine, and muscarinic acetylcholine receptors in different neuronal and glial subclasses. Certain cell subclasses correlated with known attributes of granule cells, and we combined constellation pharmacology with genetically labeled neurons to identify and characterize Purkinje cells. This study illustrates the utility of applying constellation pharmacology to classify neuronal and glial subclasses in specific anatomical regions of the brain.

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