Molecular regulation of synaptic α5-GABAA receptors

In the adult brain, the main role for γ-aminobutyric acid receptors (GABAARs) is to control neural excitability via transient synaptic and persistent tonic inhibition. These forms of inhibition are distinct and mediated by receptors located at inhibitory synapses (phasic), and by extrasynaptic receptors (tonic). Although receptors containing the α5 subunit (α5-GABAARs) are predominantly located extrasynaptically, there are also synaptic α5-GABAARs, which, due to their significant expression in the hippocampus, are thought to play an important role in learning and memory. Consequently, selective allosteric modulators of α5-GABAARs have been proposed as therapeutic options for cognitive dysfunction associated with conditions such as Down syndrome, Alzheimer’s disease, schizophrenia, and autistic spectrum disorder. However, how hippocampal synaptic inhibition via α5-GABAARs is regulated, remains largely unknown. Here, online prediction software for post-translational modification and mass-spectrometry analyses were used to discover phosphorylation sites in the intracellular domain of the α5 subunit that may regulate the subcellular location of the α5-GABAARs. To assess this, one of the key phosphorylated residues α5-S374 was mutated to phosphomimetic and phosphodead residues. Whole-cell patch clamp recordings in wild-type and mutant transfected HEK293 cells were used to reveal that α5-S374 has a significant role in receptor function and is likely to be phosphorylated by the kinase GSK3β. By using a combination of electrophysiology and structured illumination microscopy (SIM) in transfected cultured hippocampal neurons, phosphorylation of α5S374 was found to regulate the synaptic location of α5-GABAARs and thus control phasic inhibition. The underlying molecular mechanism behind modified α5-receptor trafficking is likely to be altered binding between phosphorylated α5-GABAARs and inhibitory synaptic scaffold proteins. Although several drugs can target α5-GABAARs, a greater understanding of the molecular mechanisms by which neurons control their accumulation at synaptic sites, and thus regulate phasic inhibition, is greatly needed to elucidate the role of α5-GABAARs in cognition