The Drosophila Acetylcholine Receptor Subunit Dα5 Is Part of an α-Bungarotoxin Binding Acetylcholine Receptor

Abstract

The central nervous system of Drosophila melanogaster contains an ␣-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 ␣-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 mol of 125 I-␣-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 Nicotinic acetylcholine receptors (nAChRs)1 are key elements of fast cholinergic synaptic transmission (1, 2). They are part of a large superfamily of ligand-gated ion channels comprising 5HT 3 , GABA A , and GABA C , glycine, and invertebrate anionic glutamate receptors. nAChRs are widely distributed in both vertebrates and invertebrates. In addition to their occurrence in the highly specialized tissue of the electrical organ of certain species of fish (Torpedo californica, Electrophorus electricus), nAChRs are mostly found at the vertebrate neuromuscular junction and the nervous system of both vertebrates and invertebrates (3, 4).Structurally, nAChRs are formed by assembly of their subunits into a pentameric membrane protein complex. Subunits carrying two conserved adjacent cysteines in the amino-terminal domain in the vicinity of the acetylcholine binding site are classified as ␣-subunits, while the remainder of the subunit types are designated non-␣ or -␤. The individual subunits have a large amino-terminal domain, which includes the binding site for the ligand, followed by four membrane spanning domains M1-M4. The M2 segments of the five subunits form the lining of the ion channel, which is transiently permeable to cations (Na ϩ , K ϩ , Ca 2ϩ ) upon binding of the ligand acetylcholine (ACh). Between M3 and M4 a large intracellular loop exists, which contains several phosphorylation sites required for receptor stabilization (for reviews, see Refs. 3, 5, and 6).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. Nicotinic acetylcholine receptors from muscle have an invariant subunit composition ((␣1) 2 (␤1)␦␥ (fetal) or ⑀). Neuronal nicotinic acetylcholine receptors are composed of ␣ and non-␣-subunits with a stoichoimetry of (␣) 2 (non-␣) 3 . The large number of genes encoding nAChR subunits in the nervous system can give rise to an array of functionally distinct nAChRs. Since the vertebrate nervous system also contains homomeric nAChRs (e.g. (␣ 7 ) 5 ) the entire range ...

The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

“…There is, however, evidence from studies of native nAChRs in Drosophila that Dα5 forms part of a nAChR that is sensitive to α-bungarotoxin [46], Dα6 forms part of the spinosad-sensitive nAChR [53] and that Dα7 is required for the visually-mediated cholinergic escape response [54]. …”

The nicotinic acetylcholine receptor subunits Mdα5 and Mdβ3 on autosome 1 of Musca domestica are not involved in spinosad resistance

“…The mature protein has a calculated molecular weight of 85.3 kDa and isoelectric point of 6.44. The calculated molecular weight of Mdα5 is greater than Dα5 (56.9 kDa) and Agamα5 (56.7 kDa) (Grauso et al, 2002;Jones et al, 2005;Wu et al, 2005).…”

Effects of Snake Venom Polypeptides on Central Nervous System

“…It is interesting to note that both -bungarotoxin and CTX bind to nAChR in Drosophila brain and have been used for purification as well as characterization of this receptor from insect CNS [75]. The "neuronal" (for mammals) neurotoxin -bungarotoxin produced a long-lasting competitive blockade of insect central nAChR, and this action was not dependent on membrane potential [76].…”