The Neuropeptide Orexin-A Inhibits the GABA_A Receptor by PKC and Ca²⁺/CaMKII-Dependent Phosphorylation of Its β₁ 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, 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 GABA_A receptor (GABA_AR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OX₁R and the α₁, β₁, and γ_2S subunits of GABA_AR 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 GABA_AR, 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 GABA_AR, 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/GABA_AR 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.