The Neuropeptide Orexin-A Inhibits the GABA A Receptor by PKC and Ca 2+ /CaMKII-Dependent Phosphorylation of Its β 1 Subunit

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, OX 1 R and OX 2 R. 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 A receptor (GABA A R), the brain’s main inhibitory neuroreceptor. When HEK293 cells transfected with OX 1 R and the α 1 , β 1 , and γ 2S subunits of GABA A R 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 1 subunit of GABA A R, 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 1 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 GABA A R, indicating a process in which Ox-A could promote phosphorylation of the β 1 subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABA A R 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.