Abstract
The central nervous system of Drosophila melanogaster contains an alpha-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 alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-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-alpha-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 D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.
Highlights
Nicotinic acetylcholine receptors1 are key elements of fast cholinergic synaptic transmission [1, 2]
Photoaffinity Labeling of the Acetylcholine Receptor Protein— Derivatization of 125I-␣-bungarotoxin with methyl-4-azidobenzoimidate had no noticeable effect on the ability of the toxin to bind to the membrane bound ␣-bungarotoxin-binding protein
The dissociation kinetics of the derivatized toxin by unlabeled ␣-bungarotoxin prior to photoactivation was unaffected displacing more than 80% of the derivatized toxin in 2 h
Summary
Nicotinic acetylcholine receptors (nAChRs) are key elements of fast cholinergic synaptic transmission [1, 2]. Biochemical analysis of receptors subunits composition in a variety of species as well the information obtained from the sequencing of the genome of humans, mice, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster indicates a large number of genes encoding nicotinic acetylcholine receptor subunits. Immunoprecipitation experiments indicate that the ALS and SAD subunits might be part of a receptor complex while D␣3 and ARD might be part of a different receptor subtype [17] It should be noted, that it has not been possible to reconstitute functional nAChRs from Drosophila in a heterologous system with any of these subunit combinations tested so far [18, 19]. The sequence of a peptide obtained after tryptic digestion and microbore
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