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- Title
- New Tools for Targeted Disruption of Cholinergic Synaptic Transmission in Drosophila melanogaster.
- Creator
- Mejia, Monica, Heghinian, Mari D., Mari, Frank, Godenschwege, Tanja A., McCabe, Brian D.
- Abstract/Description
-
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The a7 subtype of nAChRs is involved in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster a7 (Da7) has the closest sequence homology to the vertebrate a7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Da7 subunits are essential for the function of the Giant Fiber...
Show moreNicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The a7 subtype of nAChRs is involved in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster a7 (Da7) has the closest sequence homology to the vertebrate a7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Da7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Da7 nAChR’s ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Da7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Da7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Da7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Da7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Da7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Da7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling.
Show less - Date Issued
- 2013-05-30
- PURL
- http://purl.flvc.org/fau/fd/FAUIR000083
- Format
- Citation
- Title
- Discovery and biological characterization of conotoxins from the venom of Conus Brunneus in Drosophila Melanogaster.
- Creator
- Heghinian, Mari D., Mari, Frank, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
- Abstract/Description
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Cone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone...
Show moreCone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone snails (from either dissected or “milked” venom) is minimal, thus hindering the wide use of bioassay-guided approaches for compound discovery. Biochemical-based approaches for discovery by means of identification and characterization of venom components can be used due to their compatibility with the small quantities of cone snail venom available; however, no direct assessment of the bioactivity can be gleaned from these approaches. Therefore, newly discovered conotoxins must be acquired synthetically, which can be difficult due to their complicated folding motifs. The ability to test small quantities of peptide for bioactivity during the purification process can lead to the discovery of novel components using more direct approaches. Presented here is the description of use of an effective method of bioassay-guided fractionation for the discovery of novel alpha conotoxins as well as further biological characterization of other known alpha conotoxins. This method requires minimal amounts of sample and evaluates, via in vivo electrophysiological measurements, the effect of conotoxins on the functional outputs of a well-characterized neuronal circuit in Drosophila melanogaster known as the giant fiber system. Our approach uses reversed-phase HPLC fractions from venom dissected from the ducts of Conus brunneus in addition to synthetic alpha conotoxins. Fractions were individually tested for activity, re-fractionated, and re-tested to narrow down the compound responsible for activity. A novel alpha conotoxin, bru1b, was discovered via the aforementioned approach. It has been fully characterized in the giant fiber system through the use of mutant flies, as well as tested in Xenopus oocytes expressing nicotinic acetylcholine channels and against the acetylcholine binding protein. Other well-known alpha conotoxins have also been characterized in the giant fiber system.
Show less - Date Issued
- 2014
- PURL
- http://purl.flvc.org/fau/fd/FA00004122, http://purl.flvc.org/fau/fd/FA00004122
- Subject Headings
- Drosophila melanogaster, Gastropoda -- venom, Peptides -- Structure, Venom
- Format
- Document (PDF)