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.

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.

Blockade and Activation of the Human Neuronal Nicotinic Acetylcholine Receptors by Atracurium and Laudanosine

2001 ◽  
Vol 94 (4) ◽  
pp. 643-651 ◽  
Author(s):  
Florence Chiodini ◽  
Eric Charpantier ◽  
Dominique Muller ◽  
Edomer Tassonyi ◽  
Thomas Fuchs-Buder ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Receptors ◽  
Receptor Subtype ◽  
Xenopus Laevis Oocytes ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Neuronal Nicotinic Receptors ◽  
Arterial Blood ◽  
Low Concentrations

Background Curaremimetic nondepolarizing muscle relaxants are widely used in clinical practice to prevent muscle contraction either during surgery or during intensive care. Although primarily acting at the neuromuscular junction, these compounds can cause adverse effects, including modification of cardiac rhythm, arterial blood pressure, and in the worst cases, triggering of seizures. In this study, we assessed the interaction of atracurium and its metabolite, laudanosine, with neuronal nicotinic receptors. Methods The human neuronal nicotinic receptors alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha7 are heterologously expressed in Xenopus laevis oocytes, and the effect of atracurium and its degradation product, laudanosine, were studied on these receptors. Results Atracurium and laudanosine inhibited in the micromolar range the major brain alpha4beta2 receptor and the ganglionic alpha3beta4 or alpha3beta4alpha5 and the homomeric alpha7 receptors. For all four receptors, inhibition was rapid and readily reversible within less than 1 min. Atracurium blockade was competitive at alpha4beta2 and alpha7 receptors but displayed a noncompetitive blockade at the alpha3beta4 receptors. Inhibition at this receptor subtype was not modified by alpha5. Laudanosine was found to have a dual mode of action; first, it competes with acetylcholine and, second, it blocks the ionic pore by steric hindrance. At low concentrations, these two drugs are able to activate both the alpha4beta2 and the alpha3beta4 receptors. Conclusion Adverse effects observed during atracurium administration may be attributed, at least partly, to an interaction with neuronal nicotinic receptors.

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.

scholarly journals αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish

Toxins ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 197
Author(s):  
Matthew J. Rybin ◽  
Henrik O’Brien ◽  
Iris Bea L. Ramiro ◽  
Layla Azam ◽  
J. Michael McIntosh ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Functional Characterization ◽  
Cone Snail ◽  
Nicotinic Acetylcholine ◽  
New Class ◽  
Goldfish Carassius Auratus ◽  
Snake Neurotoxin

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 μM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

scholarly journals Functional characterization of mouse α4β2 nicotinic acetylcholine receptors stably expressed in HEK293T cells

2004 ◽  
Vol 91 (5) ◽  
pp. 1138-1150 ◽  
Author(s):  
Mark S. Karadsheh ◽  
M. Salman Shah ◽  
Xin Tang ◽  
Robert L. Macdonald ◽  
Jerry A. Stitzel
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Functional Characterization ◽  
Nicotinic Acetylcholine

Zinc Potentiates Neuronal Nicotinic Receptors by Increasing Burst Duration

2008 ◽  
Vol 99 (2) ◽  
pp. 999-1007 ◽  
Author(s):  
Bernard Hsiao ◽  
Karla B. Mihalak ◽  
Karl L. Magleby ◽  
Charles W. Luetje
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Large Fraction ◽  
Burst Duration ◽  
Dependent Manner ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Open Probability ◽  
Neuronal Nicotinic Receptors ◽  
Channel Current

Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated α4β4 or α4β2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 μM) activated α4β4 receptors in outside-out patches were similar in the absence and the presence of 100 μM zinc (3.0 ± 0.1 and 2.9 ± 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined α4β4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 ± 0.002) was increased 4.5-fold by 100 μM zinc (0.050 ± 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 ± 38 ms in the absence of zinc to 830 ± 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.

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