Sachidanandan Divya, Reddy Haritha P, Mani Anitha, Hyde Geoffrey J, Bera Amal Kanti
Department of Neurophysiology, Institute of Physiology, University of Würzburg, Röntgenring 9, 97070, Würzburg, Germany.
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Sardar Patel Road, Chennai, Tamil Nadu, 600036, India.
J Mol Neurosci. 2017 Apr;61(4):459-467. doi: 10.1007/s12031-017-0886-0. Epub 2017 Jan 19.
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, OXR and OXR. 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 GABA receptor (GABAR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OXR and the α, β, and γ subunits of GABAR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the β subunit of GABAR, 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 β 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 GABAR, indicating a process in which Ox-A could promote phosphorylation of the β subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABAR 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.
食欲素-A和食欲素-B（Ox-A，Ox-B）是由少数起源于下丘脑并广泛投射至大脑的神经元产生的神经肽。食欲素在1998年才被发现，目前已知它能调节多种行为。最显著的是，它们有助于稳定清醒状态，这一作用在发作性睡病和失眠的临床治疗中具有重要意义。食欲素与两种亚型的G蛋白偶联受体（主要是突触后受体），即OXR1和OXR2结合。Ox-A与OXR1结合的主要作用是通过细胞膜阳离子通道介导直接的去极化影响，但各种各样的突触前和突触后的次要作用也逐渐显现。鉴于抑制性γ-氨基丁酸能神经元也会影响食欲素调节的行为，预计这两种系统之间会发生相互作用，但在细胞水平上，人们了解甚少，可能的机制仍不明晰。在此，我们采用表达系统方法来研究Ox-A与大脑主要抑制性神经受体γ-氨基丁酸受体（GABAR）之间可能存在的突触后相互作用的可行性及性质。当用OXR1以及GABAR的α、β和γ亚基转染的人胚肾293（HEK293）细胞暴露于Ox-A时，γ-氨基丁酸诱导的电流受到抑制，且呈钙依赖性。这种抑制与GABAR的β亚基磷酸化增加有关，并且这种抑制本身可被（1）蛋白激酶C和钙调蛋白激酶II的激酶抑制剂以及（2）β亚基丝氨酸409突变为丙氨酸所减弱，丝氨酸409是先前在其他途径的磷酸化依赖性调节中确定的位点。这些结果首次直接支持了Ox-A与GABAR之间突触后相互作用的可行性，表明存在这样一个过程，即Ox-A可促进β亚基的磷酸化，减少γ-氨基丁酸诱导的超极化电流。在这个模型中，Ox-A/GABAR相互作用会使Ox-A的去极化影响增强，这是一种正反馈，例如可能有助于突然觉醒的能力。
