Cross-linking of 125I-α-bungarotoxin to Drosophila head membranes identifies a 42 kDa toxin binding polypeptide

doi:10.1016/0304-3940(92)90204-K

Neuroscience Letters

Volume 145, Issue 1, 28 September 1992, Pages 63-66

https://doi.org/10.1016/0304-3940(92)90204-K Get rights and content

Abstract

The nicotinic acetylcholine receptor (nAChR) antagonist α-bungarotoxin (α-Btx) binds to two different classes of high affinity binding sites from the Drosophila central nervous system. We have used the bivalent reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to cross-link ¹²⁵I-α-Btx (M_r=8 kDa) to Drosophila head membranes. Upon sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), on adduct of M_r ≈50 kDa was identified, suggesting that a 42 kDa polypeptide binds the toxin. Adduct formation was inhibited by other ch ligands. Detergent-solubilized receptor complexes containing the cross-linked products were immunoprecipitated by antisera against two nAChR subunits previously identified by molecular cloning, the ALS and ARD proteins, suggesting that the 42 kDa toxin binding polypeptide constitutes a component of the previously described class 1 α-Btx binding site.

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The Drosophila acetylcholine receptor subunit Dα5 is part of an α-bungarotoxin binding acetylcholine receptor
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Citation Excerpt :
The results of our photoaffinity labeling experiments reveal two major bands of 42 and 57 kDa with additional weakly labeled bands of 102, 68, and 26 kDa. Using chemical cross-linking of 125I-α-bungarotoxin with a carbodiimide Schloss et al. (20) detected a polypeptide of 42 kDa. Tomizawa et al. (22) reported the specific labeling of a 66-kDa polypeptide with [3H]azidoneonicotinoid.
The central nervous system of Drosophila melanogaster contains an α-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 m sodium chloride and then purified using an α-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 μmol of ¹²⁵I-α-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of ¹²⁵I-α-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H₂O and D₂O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s_20,_w of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila Dα5 subunit by sequence comparison. A peptide-specific antibody raised against the Dα5 subunit provides further evidence that this subunit is a component of an α-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.
[<sup>125</sup>I]Azidonicotinoid photoaffinity labeling of insecticide-binding subunit of Drosophila nicotinic acetylcholine receptor
1997, Neuroscience Letters
The novel synthetic nicotinoid insecticide imidacloprid is a high affinity ligand for the insect nicotinic acetylcholine receptor (nAChR). The goal of this study is to identify the ligand- and insecticide-binding subunit of Drosophila nAChR with a novel [¹²⁵I]azidonicotinoid ([¹²⁵I]AN) photoaffinity probe modeled on imidacloprid. [¹²⁵I]AN photoaffinity labels a single polypeptide in Drosophila head membranes corresponding in molecular mass to 66 kDa at a specific site inhibited by various cholinergic ligands including (−)-nicotine, cytisine, carbachol, α-bungarotoxin and d-tubocurarine as well as the insecticides imidacloprid and acetamiprid, pharmacologically consistent with the ligand- and insecticide-binding subunit. The Drosophila nAChR, isolated with three putative subunits (69, 66 and 61 kDa) using a nicotinoid-agarose affinity column, is labeled by [¹²⁵I]AN primarily at the 66 kDa subunit and secondarily at the 61 kDa subunit. Clearly, the novel synthetic nicotinoid insecticides are valuable contributors in exploring the structure and function of the Drosophila nAChR.
Immunocytochemical detection of the serotonin transporter in rat brain
1996, Neuroscience
The regional distribution of the serotonin uptake system was studied in rat brain using a specific polyclonal antibody raised against the putative extracellular loop between transmembrane domains 7 and 8 of the cloned rat serotonin transporter. Light microscope analysis with fluorescence and avidin-biotin-peroxidase techniques revealed a punctate staining as well as numerous labelled thin fibres, which exhibited accumulation of reaction end-product deposit over varicosities. These immunopositive processes were widely and heterogeneously distributed in the rat brain. High densities of immunoreactivity were seen within the caudate-putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, Islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nucleus and olfactory nuclei. We also found strong expression of serotonin transporter in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum moleculare of CAI-CA3 regions of the hippocampus. Within the raphe nuclei, a moderate to high incidence of stained processes was observed, and immunopositive cell bodies were detected in the dorsal raphe nucleus. In addition, some immunoreactive fibres were present in the molecular and granular layers of the cerebellum as well as in the cochlear and olivary nuclei. In none of the regions analysed was evidence for glial staining obtained.
The present immunocytochemical data reveal a widespread and heterogeneous distribution of the serotonin transporter in rat brain and suggest that serotonin transporter is preferentially sorted into axons, where it appears concentrated at varicosities and terminal boutons.
Acetylcholine receptors of the Drosophila brain: A 900 bp promoter fragment contains the essential information for specific expression of the ard gene in vivo
1994, FEBS Letters
Theard gene encodes a β-subunit ofDrosophila nicotinic acetylcholine receptors specifically expressed in a subset of neurons. To identify thecis-regulatory region responsible for this cell-specific expression, various 5′ fragments of theard gene were fused to alacZ reporter gene and introduced into theDrosophila genome. A DNA fragment spanning ∼760 bp upstream and ∼140 bp downstream of a cluster of putative transcription start sites produced a pattern of β-galactosidase activity that resembles the distribution of ARD transcripts. Both in embryos and adults the levels oflacZ RNA were similar to those of endogenous ARD transcripts, suggesting that the 900 bp fragment harbors all essential elements for proper expression of theard gene.
Nicotinic acetylcholine receptors of the central nervous system of Drosophila
1992, BBA - Molecular Cell Research
Dβ3, an atypical nicotinic acetylcholine receptor subunit from Drosophila: Molecular cloning, heterologous expression and coassembly
2002, Journal of Neurochemistry

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Neuroscience Letters

Abstract

References (19)

Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors

Trends Pharmacol. Sci.

Nicotinic receptors in the brain of Drosophila melanogaster demonstrated by autoradiography with [¹²⁵I]-α-bungarotoxin

Brain Res.

Structure and developmental expression of the Dα2 gene encoding a novel nicotinic acetylcholine receptor protein of Drosophila melanogaster

FEBS Lett.

Photochemical cross-linking of cell membranes

J. Biol. Chem.

Is the insect neuronal nAChR the ancestral receptor protein?

Trends Neurosci.

SBD, a novel structural subunit of the Drosophila nicotinic acetylcholine receptor, shares its genomic localization with two α-subunits

FEBS Lett.

Cross-linking the major proteins of the isolated erythrocyte membrane

J. Mol. Biol.

Characterization of the α-bungarotoxin receptor in chickembryo retina

Eur. J. Biochem.

Conversion of neural nicotinic acetylcholine receptors from Drosophila to vertebrate nervous system

EMBO J.

Cited by (16)

The Drosophila acetylcholine receptor subunit Dα5 is part of an α-bungarotoxin binding acetylcholine receptor

[<sup>125</sup>I]Azidonicotinoid photoaffinity labeling of insecticide-binding subunit of Drosophila nicotinic acetylcholine receptor

Immunocytochemical detection of the serotonin transporter in rat brain

Acetylcholine receptors of the Drosophila brain: A 900 bp promoter fragment contains the essential information for specific expression of the ard gene in vivo

Nicotinic acetylcholine receptors of the central nervous system of Drosophila

Dβ3, an atypical nicotinic acetylcholine receptor subunit from Drosophila: Molecular cloning, heterologous expression and coassembly