title: The Neuropeptide Orexin-A Inhibits the GABAAReceptor by PKC and Ca2+/CaMKII-Dependent Phosphorylation of Its β1Subunit.
abstract: Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OX1R and OX2R. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABAAreceptor (GABAAR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OX1R and the α1, β1, and γ2Ssubunits of GABAAR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the β1subunit of GABAAR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the β1subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABAAR, indicating a process in which Ox-A could promote phosphorylation of the β1subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABAAR crosstalk would cause the depolarising influence of Ox-A to be boosted, a type of positive feedback that could, for example, facilitate the ability to abruptly awake.
authors.affiliation: Department of Neurophysiology, Institute of Physiology, University of Würzburg, Röntgenring 9, 97070, Würzburg, Germany.
authors.forename: Divya
authors.initials: D
authors.lastname: Sachidanandan
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Sardar Patel Road, Chennai, Tamil Nadu, 600036, India.
authors.forename: Haritha P
authors.initials: HP
authors.lastname: Reddy
authors.affiliation: Life Science Division, AU-KBC Research Centre, MIT Campus, Anna University, Chromepet, Chennai, Tamil Nadu, 600044, India.
authors.forename: Anitha
authors.initials: A
authors.lastname: Mani
authors.affiliation: Write About Research, 14 Randwick Street, Randwick, Sydney, 2031, Australia.
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Hyde
authors.affiliation: Department of Neurophysiology, Institute of Physiology, University of Würzburg, Röntgenring 9, 97070, Würzburg, Germany. amal@iitm.ac.in.
authors.forename: Amal Kanti
authors.initials: AK
authors.lastname: Bera
mesh_terms: D000200:Action Potentials; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D064446:Orexin Receptors / Q000235:genetics / Q000378:metabolism; D000068797:Orexins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*
chemical_list: C578630:HCRTR1 protein, human; C578626:HCRTR2 protein, human; D064446:Orexin Receptors; D000068797:Orexins; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium
keywords: GABA; Orexin; Phosphorylation; Protein kinase; Sleep-wake cycle