γ-Aminobutyric acid type A receptors (GABA_ARs) are anion permeable channels that mediate the majority of fast neuronal inhibition in the adult mammalian brain. Tonic inhibition is a specific form of inhibitory current characterized by a constant reduction in the neuron's resting membrane potential, reducing the probability of action potential initiation. The GABA _AR subtypes that mediate tonic inhibition in the hippocampus are hetero-pentamers that are assembled largely from α₄β_2/3 with or without the δ subunit. To date, the endogenous mechanisms neurons use to regulate tonic inhibition remain unknown. To address this issue we have begun to analyze the role of phosphorylation on GABA_ARs containing α ₄. Here we reveal that the α₄ subunit is phosphorylated at serine 443 (Ser⁴⁴³) by protein kinase C (PKC). We further demonstrate that PKC activity increases the cell surface expression of α ₄ in the hippocampus by enhancing the protein's stability within the endoplasmic reticulum, thereby increasing the rate of insertion into the plasma membrane. We also show that PKC activity is capable of increasing α ₄ subunit-mediated tonic current by preventing current rundown.

In addition, we analyzed the effects of neurosteroids, one of the most potent endogenous modulators of GABAergic inhibition, on α₄ subunit activity. Here we reveal that tetrahydroxycorticosterone (THDOC) treatment results in increased phosphorylation of α₄ and a concomitant increase in α₄ cell surface expression. We further demonstrate that this effect is mediated by PKC and is dependent on the Ser⁴⁴³ residue in α₄.

We have also begun to analyze the mechanisms involved in recruiting PKC to GABA_AR subunits. We know that PKC has a high affinity for the β ₃ subunit. Here we will demonstrate the effect of reducing this affinity on α₄ activity. Expressing a phospho-mutant version of β ₃, ^βS408/9A, known to not bind PKC, results in the loss of phosphorylation and reduction in cell surface expression of the α ₄ subunit. Finally, we examined the behavioral effects of this mutant in a knock-in mouse expressing β₃^S408/9A. In this study, we show that the mutant displays higher levels of asocial-like and anxiety-like behavior, two abnormalities that have been linked to alterations in α₄ subunit-mediated neuronal activity.