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 GABAA receptor (GABAAR), the brain’s main inhibitory neuroreceptor. When HEK293 cells transfected with OX1R and the α1, β1, and γ2S subunits 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 β1 subunit 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 β1 subunit, 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 β1 subunit, 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.
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Acknowledgements
We express our sincere gratitude to Dr. Takeshi Sakurai and Dr. Neil Harrison for providing the orexin receptor and GABA receptor clones, respectively. We are also grateful to Sucheta Sridhar for her critical comments on the manuscript. D.S. and H.P.R. are thankful to IIT Madras and the Department of Biotechnology, India (DBT), respectively, for the fellowships provided during the course of their PhD studies. The study was supported by the Council of Scientific and Industrial Research (CSIR), India.
Author Contributions
D.S. and A.K.B. conceived the idea, analysed the data, and wrote the manuscript. D.S., H.P.R., and A.M. performed the experiments. G.J.H. analysed the data and wrote the manuscript.
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Sachidanandan, D., Reddy, H.P., Mani, A. et al. The Neuropeptide Orexin-A Inhibits the GABAA Receptor by PKC and Ca2+/CaMKII-Dependent Phosphorylation of Its β1 Subunit. J Mol Neurosci 61, 459–467 (2017). https://doi.org/10.1007/s12031-017-0886-0
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DOI: https://doi.org/10.1007/s12031-017-0886-0