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1.
Elife ; 3: e01473, 2014.
Article in English | MEDLINE | ID: mdl-24497543

ABSTRACT

In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light-dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep. DOI: http://dx.doi.org/10.7554/eLife.01473.001.


Subject(s)
Drosophila Proteins/metabolism , Receptors, Nicotinic/metabolism , Sleep , Animals , Circadian Rhythm , Drosophila Proteins/genetics , Drosophila melanogaster , Gene Expression Regulation , Genotype , Homeostasis , Mutation , Phenotype , Photoperiod , Receptors, Nicotinic/genetics , Signal Transduction , Sleep/genetics , Time Factors
2.
Mol Pharmacol ; 79(1): 126-40, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20923852

ABSTRACT

α6ß2ß3* acetylcholine receptors (AChRs) on dopaminergic neurons are important targets for drugs to treat nicotine addiction and Parkinson's disease. However, it has not been possible to efficiently express functional α6ß2ß3* AChRs in oocytes or transfected cells. α6/α3 subunit chimeras permit expression of functional AChRs and reveal that parts of the α6 M1 transmembrane domain and large cytoplasmic domain impair assembly. Concatameric subunits permit assembly of functional α6ß2ß3* AChRs with defined subunit compositions and subunit orders. Assembly of accessory subunits is limiting in formation of mature AChRs. A single linker between the ß3 accessory subunit and an α4 or α6 subunit is sufficient to permit assembly of complex ß3-(α4ß2)(α6ß2) or ß3-(α6ß2)(α4ß2) AChRs. Concatameric pentamers such as ß3-α6-ß2-α4-ß2 have been functionally characterized. α6ß2ß3* AChRs are sensitive to activation by drugs used for smoking cessation therapy (nicotine, varenicline, and cytisine) and by sazetidine. All these are partial agonists. (α6ß2)(α4ß2)ß3 AChRs are most sensitive to agonists. (α6ß2)2ß3 AChRs have the greatest Ca²+ permeability. (α4ß2)(α6ß2)ß3 AChRs are most efficiently transported to the cell surface, whereas (α6ß2)2ß3 AChRs are the least efficiently transported. Dopaminergic neurons may have special chaperones for assembling accessory subunits with α6 subunits and for transporting (α6ß2)2ß3 AChRs to the cell surface. Concatameric pentamers and pentamers formed from combinations of trimers, dimers, and monomers exhibit similar properties, indicating that the linkers between subunits do not alter their functional properties. For the first time, these concatamers allow analysis of functional properties of α6ß2ß3* AChRs. These concatamers should enable selection of drugs specific for α6ß2ß3* AChRs.


Subject(s)
Chimera/genetics , Protein Subunits/genetics , Receptors, Nicotinic/biosynthesis , Receptors, Nicotinic/genetics , Animals , Chimera/metabolism , Female , Gene Expression Regulation , Humans , Oocytes , Protein Subunits/biosynthesis , Torpedo/genetics , Xenopus laevis
3.
Mol Pharmacol ; 74(1): 132-43, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18381563

ABSTRACT

Accessory subunits in heteromeric nicotinic receptors (AChRs) do not take part in forming ACh binding sites. alpha5 and beta3 subunits can function only as accessory subunits. We show that both alpha5 and beta3 efficiently assemble in human alpha4beta2(*) AChRs expressed in permanently transfected human embryonic kidney (HEK) cell lines. Only (alpha4beta2)(2)alpha5, not (alpha4beta2)(2)beta3 AChRs, have been detected in brain. The alpha4beta2alpha5 line expressed 40% more AChRs than the parent alpha4beta2 line and was equally sensitive to up-regulation by nicotine. The alpha4beta2beta3 line expressed 25-fold more AChRs than the parental line and could not be further up-regulated by nicotine. Relative sensitivity to activation by ACh depends on the accessory subunit, beta2 conferring the greatest sensitivity, alpha5 less, and beta3 and alpha4 much less. Accessory subunits form binding sites for positive allosteric modulators, as illustrated by the observation that alpha5 conferred high sensitivity to galanthamine. In the presence of alpha5 or beta3, stable, partially degraded, dead end intermediates accumulated within the cells. These may have the form alpha5alpha4beta2alpha5. The efficiency with which alpha5 and beta3 assemble with alpha4 and beta2 and the necessity of avoiding formation of potentially toxic intermediates may explain why alpha5 and beta3 seem to be transcribed at low levels in brain. Autosomal dominant nocturnal frontal lobe epilepsy can be caused by the alpha4 mutation S247F. This mutant did not produce functional AChRs unless cells were cotransfected with alpha5, beta3, or alpha6 to replace alpha4 as accessory subunit.


Subject(s)
Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/metabolism , Up-Regulation/drug effects , Acetylcholine/pharmacology , Allosteric Regulation , Antibodies, Monoclonal/metabolism , Binding Sites , Biotinylation , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Cell Line , Galantamine/pharmacology , Humans , Inhibitory Concentration 50 , Kidney/cytology , Mecamylamine/pharmacology , Mutation , Nicotine/metabolism , Pyridines/metabolism , Radioimmunoassay , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Transfection
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