Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems (original) (raw)
Related papers
European Journal of Biochemistry, 1990
A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) a-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR a-subunit. The insect cell-expressed a-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1 -2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor a-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR a-subunit and carried the binding site for the snake venom toxin, a-bungarotoxin. Bound a-bungarotoxin was displaced in competition binding assays by a-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR a-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound a-bungarotoxin.
Activation of nicotinic acetylcholine receptors on cultured Drosophila and other insect neurones
The Journal of physiology, 1993
1. Using whole-cell and single channel recordings, we have examined the properties of acetylcholine (ACh)-activated currents in neurones from larval and pupal Drosophila melanogaster (fruit fly), larval and embryonic Musca domestica (house fly), and nymphal Schistocerca gregaria (locust). 2. In all preparations, single channel recordings revealed two major classes of ACh-activated channels, with average conductances of approximately 32 and 59 pS. 3. At ACh concentrations from 1 to 10 microM, channel activity in Drosophila larval neurones occurs in bursts with an average of 1-2 openings. Open times and burst durations are described by one or two exponentials. Burst durations for the 32 pS channel (approximately 3 ms, slow component) were longer than those for the 59 pS channel (approximately 1.0 ms). The mean open interval duration for the 32 pS channel (slow component) was also longer than that of the 59 pS channel. 4. At high ACh (20-200 microM) concentrations, bursts of the smalle...
Exploring the pharmacological properties of insect nicotinic acetylcholine receptors
Trends in Pharmacological Sciences, 2007
Glossary a-Bungarotoxin (a-Bgt): toxin from snake venom. a-Bgt binding is considered to represent the distribution of a7-subunit-containing nACh receptors. DEG: degeneration of certain neurons. In Caenorhabditis elegans, there are 42 different nACh receptor subunits, including the deg-3 group. des-2 is another gene in this group. DES: degeneration suppressor. Mutations in the gene encoding this protein suppress the degeneration caused by deg-3. Drosophila Da7 mutant: excisions of P elements in the Da7 subunits lead to several alleles. nACh receptor subtype: a specific combination of identical (homomeric) or different (heteromeric) subunits that forms a pentameric nACh receptor.
Molecular pharmacology, 2001
Although molecular biology provides new insights into the subunit compositions and the stoichiometries of insect neuronal nicotinic acetylcholine receptors (nAChRs), our knowledge about the phosphorylation/dephosphorylation mechanisms of native neuronal nAChRs is limited. The regulation of alpha-bungarotoxin-resistant nAChRs was studied on dissociated adult dorsal unpaired median neurons isolated from the terminal abdominal ganglion of the cockroach Periplaneta americana, using whole-cell, patch-clamp technique. Under 0.5 microM alpha-bungarotoxin treatment, pressure ejection application of nicotine or acetylcholine onto the cell body induced an inward current exhibiting a biphasic current-voltage relationship. We found that two distinct components underlying the biphasic curve differed in their ionic permeability and pharmacology (one being sensitive to d-tubocurarine, and the other affected only by mecamylamine and alpha-conotoxin ImI). This indicated that two types of alpha-bunga...
Neurochemistry International, 1990
A~tract--In this report evidence is presented for a membrane-associated polypeptide that regulates ligand binding properties of a neuronal acetylcholine receptor. Removal of membrane-associated compounds reversibly increased the number of BGTX binding sites and decreased the number of binding sites for ACh in neuronal membranes, suggesting the existence of endogenous membrane-associated factors that might allosterically modulate the ligand binding sites of receptor protein. The regulatory factor was purified and identified as 20 kDa polypeptide. The purified polypeptide was found to be phosphorylated by cAMPdependent protein kinase, which caused inactivation of the modulatory polypeptide and thus might control their function.
J Neurochem, 2002
Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two ␣ subunits, ␣-like subunit (ALS) and Drosophila ␣2 subunit (D␣2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one ␣ subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate D␣2, and vice versa, and thereby suggest that these two ␣ subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/2 or D␣2/2) and ternary (ALS/D␣2/2) receptor complexes was made on the basis of their dose -response curve to acetylcholine as well as their sensitivity to ␣-bungarotoxin or dihydro--erythroidine. These data demonstrate that the presence of the two ␣ subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.
Journal of Neurochemistry, 2002
The second b-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced speci®c antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the a-subunit ALS in the synaptic neuropil. The subunits ALS, Da2 and SBD can be co-puri®ed by a-bungarotoxin af®nity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and Da2 and, vice versa, ALS and Da2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaf®nity chromatography with immobilized antibodies against ALS and Da2 revealed the existence of nAChR complexes that include ALS, Da2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional b-subunit, but neither ALS nor Da2 nor the third a-subunit Da3. These ®ndings suggest a considerable complexity of the Drosophila nicotinic receptor system. . 2 These authors contributed equally to this work.
Neuroscience, 1987
A panel of monoclonal antibodies with known specificity for the well-characterized nicotinic acetylcholine receptor from the electroplax of Torpedo californicu, many of which cross-react with the mammalian muscle acetylcholine receptor, were examined for cross-reactivity in the fly, Drosophila melunoguster. Monoclonal antibodies with specificities for different epitopes on the transmembrane receptor complex from Torpedo cross-react with different regional subsets of neural tissue in Drosophila. Axonal tracts, neuropil, mechano-sensory bristle elements and photoreceptors, each are detected by separate monoclonal antibody classes corresponding to different epitope domains. A preliminary characterization of an antigenic determinant in Drosophila heads recognized by one of the cross-reacting monoclonal antibodies is presented. Monoclonal antibodies such as these may be useful in identifying molecules of homologous structure or function, possibly including a neuronal acetylcholine receptor.
Gene, 2005
Acetylcholine is the principal excitatory neurotransmitter in the central nervous system of insects. Nicotinic acetylcholine receptors, which belong to the ligand-gated ion channel family, constitute important targets for insecticides. In the honeybee Apis mellifera, pharmacological evidence supports the existence of several nicotinic acetylcholine receptors. In this paper, we report the identification of three new genes that encode nicotinic acetylcholine receptor a-subunits in the honeybee. Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support their classification as Apisa2, Apisa7-1 and Apisa7-2 subunits. Based on in situ hybridization experiments, we determined their expression patterns in the different brain regions of pupae and adult honeybees. Our results show that these nicotinic acetylcholine receptor subunits are differently expressed among the brain regions and that they appear at different stages of honeybee development. D