Interactions of charatoxins and nereistoxin with the nicotinic acetylcholine receptors of insect cns andTorpedo electric organ

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

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Use of Monoclonal Antibodies to Study the Structure and Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

10.21236/ada198425 ◽

1988 ◽

Author(s):

Jon M. Lindstrom

Keyword(s):

Monoclonal Antibodies ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Electric Organ ◽

Structure And Function ◽

Nicotinic Acetylcholine ◽

And Function

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Effects of Bis(7)-Tacrine on Spontaneous Synaptic Activity and on the Nicotinic ACh Receptor of Torpedo Electric Organ

Journal of Neurophysiology ◽

10.1152/jn.2001.86.1.183 ◽

2001 ◽

Vol 86 (1) ◽

pp. 183-189 ◽

Cited By ~ 16

Author(s):

Esteve Ros ◽

Jordi Aleu ◽

Inmaculada Gomez De Aranda ◽

Carles Cantí ◽

Yuan-Ping Pang ◽

...

Keyword(s):

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Electric Organ ◽

Synaptic Activity ◽

Hill Coefficient ◽

Nicotinic Acetylcholine ◽

Torpedo Electric Organ ◽

Induced Currents

Bis(7)-tacrine is a potent acetylcholinesterase inhibitor in which two tacrine molecules are linked by a heptylene chain. We tested the effects of bis(7)-tacrine on the spontaneous synaptic activity. Miniature endplate potentials (MEPPs) were recorded extracellularly on slices of electric organ of Torpedo marmorata. Bis(7)-tacrine, at a concentration of 100 nM, increased the magnitudes that describe MEPPs: amplitude, area, rise time, rate of rise, and half-width. We also tested the effect of bis(7)-tacrine on nicotinic acetylcholine receptors by analyzing the currents elicited by acetylcholine (100 μM) in Torpedo electric organ membranes transplanted in Xenopus laevis oocytes. Bis(7)-tacrine inhibited the acetylcholine-induced currents in a reversible manner (IC50 = 162 nM). The inhibition of nicotinic acetylcholine receptors was not voltage dependent, and bis(7)-tacrine increased the desensitization of nicotinic acetylcholine receptors. The Hill coefficient for bis(7)-tacrine was −0.72 ± 0.02, indicating that bis(7)-tacrine binds to the nicotinic acetylcholine receptor in a molecular ratio of 1:1, but does not affect the binding of α-bungarotoxin with the nicotinic acetylcholine receptor. In conclusion, bis(7)-tacrine greatly increases the spontaneous quantal release from peripheral cholinergic terminals at a much lower concentration than tacrine. Bis(7)-tacrine also blocks acetylcholine-induced currents of Torpedo electric organ, although the mechanism is different from that of tacrine: bis(7)-tacrine enhances desensitization, whereas tacrine reduces it.

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Genes encoding neuronal nicotinic acetylcholine receptors.

Clinical Chemistry ◽

10.1093/clinchem/35.5.731 ◽

1989 ◽

Vol 35 (5) ◽

pp. 731-737 ◽

Cited By ~ 21

Author(s):

E S Deneris ◽

J Boulter ◽

J Connolly ◽

E Wada ◽

K Wada ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Electric Organ ◽

Wide Distribution ◽

Xenopus Laevis Oocytes ◽

Neuronal Nicotinic Acetylcholine Receptors ◽

Receptor System ◽

Nicotinic Acetylcholine ◽

Beta 2 ◽

The Brain

Abstract Four genes (alpha 2, alpha 3, alpha 4, and beta 2), which encode proteins homologous to the Torpedo electric organ and vertebrate muscle nicotinic acetylcholine receptors, have been identified by cloning rat brain cDNAs. Injection of transcripts derived from these cDNAs into Xenopus laevis oocytes results in the formation of three nicotinic acetylcholine receptors. Two of these receptors, alpha 3/beta 2 and alpha 4/beta 2, have the characteristics of ganglionic nicotinic receptors. The third (alpha 2/beta 2) exhibits a previously undescribed pharmacology and thus represents a novel subtype that may be expressed in the brain. The wide distribution of alpha 2, alpha 3, alpha 4, and beta 2 transcripts in the brain indicates that neuronal nicotinic acetylcholine receptors are a major neurotransmitter receptor system.

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Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1982.0040 ◽

1982 ◽

Vol 215 (1199) ◽

pp. 241-246 ◽

Cited By ~ 115

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Messenger Rna ◽

Acetylcholine Receptors ◽

Nicotinic Receptors ◽

Xenopus Oocytes ◽

Electric Organ ◽

Nicotinic Acetylcholine ◽

Ionic Permeability ◽

Oocyte Membrane

Messenger RNA extracted from the electric organ of Torpedo was injected into Xenopus oocytes. This led to the synthesis and incorporation of functional acetylcholine receptors into the membrane of the oocyte. When activated by acetylcholine these Torpedo acetylcholine receptors in the oocyte membrane opened channels whose ionic permeability resembled that of nicotinic receptors in other cells.

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