Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila (original) (raw)
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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.
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.
FEBS Letters, 2000
DK K3 is a functional K K-subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here, we produced DK K3-specific antibodies to study which other nAChR subunits can co-assemble with DK K3 in receptor complexes of the Drosophila nervous system. Immunohistochemical studies revealed that DK K3 is co-distributed with the L L-subunit ARD in synaptic neuropil regions of the optic lobe. Both subunits can be co-purified by K K-bungarotoxin affinity chromatography. DK K3 antibodies co-immunoprecipitate DK K3 and ARD proteins and, vice versa, anti-ARD antibodies co-precipitate ARD and DK K3. These data demonstrate that one type of fly nAChRs includes these two subunits as integral components. ß
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...
Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems
Neurochemical research, 2000
Different mammalian and insect somatic host cell systems were tested in their ability to express, fold, and assemble alpha7-type neuronal acetylcholine receptor (AChR) both at the transcriptional and translational level. For this purpose we employed clonal cell lines derived from the neural crest, such as PC12 cells from a rat adrenal pheochromocytoma, and GH3 cells isolated from a rat pituitary tumor, as well as non-neuronal cells such as NIH-3T3 fibroblasts from embryonic NIH Swiss mouse and Sf9 cells from ovary tissue of the Spodoptera frugiperda butterfly. Total RNA, isolated from either transfected or non-transfected PC12, GH3 or 3T3 cells, or recombinant AcNPV-infected and mock-infected Sf9 cells was analyzed by Northern blot. PC12 cells, which endogenously express alpha7 AChR, and all its heterologous alpha7-transfectant clones, exhibited variable but generally high amounts of a single transcript. GH3 and NIH-3T3 transfectant clones and recombinant AcNPV-infected Sf9 cells ex...
eLife, 2022
Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs. Editor's evaluation The authors employ genetic and biochemical approaches to demonstrate the insecticidal effects of a snake peptide toxins. Intriguingly, they show that it targets different nicotinic acetylcholine receptor subunits than a previously identified insecticidal spider toxin. Especially their clever combination of detergent-free membrane protein extraction and mass spectrometry will no doubt prove useful to study native receptor-ligand interactions in the future.
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
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.
Neuroscience & Biobehavioral Reviews, 2012
Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system of insects where they supply fast synaptic excitatory transmission and represent a major target for several insecticides. The unbalance is striking between the abundant literature on nAChR sensitivity to insecticides and the rarity of information regarding their molecular properties and cognitive functions. The recent advent of genome sequencing disclosed that nAChR gene families of insects are rather small-sized compared to vertebrates. Behavioral experiments performed in the honeybee demonstrated that a subpopulation of nAChRs sensitive to the venom ␣-bungarotoxin and permeant to calcium is necessary for the formation of long-term memory. Concomitant data in Drosophila reported that repetitive exposure to nicotine results in a calcium-dependent plasticity of the nAChR-mediated response involving cAMP signaling cascades and indicated that ACh-induced Ca ++ currents are modulated by monoamines involved in aversive and appetitive learning. As in vertebrates, in which glutamate and NMDA-type glutamate receptors are involved in experience-associated synaptic plasticity and memory formation, insects could display a comparable system based on ACh and ␣-Bgt-sensitive nAChRs.