Neurosteroid-dependent modulation of GABAA receptors: a role for β subunit phosphorylation

Joanna Adams, Trevor G. Smart

University College London, London, UK

GABAA receptors are responsible for the majority of inhibitory synaptic transmission in the brain. Endogenous agents that can modulate GABAA receptor function will therefore have important consequences for neural activity. Neurosteroids and protein kinases are among the most potent modulators of the GABAA receptor, which, when acting individually, can enhance or depress receptor function depending on the type of neurosteroid or kinase present and the subunit combination of the receptor (Belelli et al., 2005; Moss et al., 1996). However, in vivo, these agents cannot be treated as discrete modulators of the GABAA receptor as it is likely that they will act in concert to achieve a precise fine-tuning of inhibitory neurotransmission. Therefore, the functional relationship between these two modulators, at the GABAA receptor, was investigated.

Whole cell patch clamp electrophysiology using HEK293 cells expressing recombinant α1β3γ2L GABAA receptors was used in order to determine the effects of phosphorylation, by protein kinases, on the potentiation of responses to EC20 GABA by the endogenously-produced neurosteroid, tetrahydrodeoxycorticosterone (THDOC). The potentiation of the GABA-activated response by 50nM THDOC, measured in each cell, was normalised to 100%. In the same cells, the level of potentiation induced by 50nM THDOC was significantly reduced by 55 ± 9% (mean ± s.e.m., P<0.05, paired t-test; n = 5) after treatment with 200nM staurosporine, a broad-spectrum kinase inhibitor. A similar reduction in the potentiation of 41 ± 6% (P<0.05; n = 3) was observed using 500nM bisindolylmaleimide I, a selective inhibitor of PKC. By contrast, the THDOC potentiation remained unaffected by perfusing transfected HEK cells with a myristoylated peptide inhibitor of PKA (PKI (14-22) amide). The THDOC potentiation of GABA responses was also increased by 59 ± 15% (P<0.05, n = 7) in the presence of 100nM of the PKC activator, PMA, whereas activation of PKA by internally-applied cAMP (300 μM) had no effect.

This indicates a role for PKC in setting the limits of potentiation of GABAA receptor function by neurosteroids. PKC can phosphorylate GABAA receptor β3 subunits on Serines 408 and 409 (McDonald et al., 1998). We therefore evaluated neurosteroid potentiation in cells expressing α1β3S408A,S409Aγ2L GABAA receptors. In contrast to wild-type GABAA receptors, mutant α1β3S408A,S409Aγ2L receptors showed no change in the magnitude of potentiation elicited by 50nM THDOC when treated with either staurosporine or PMA. Overall, our results suggest that phosphorylation of the GABAA receptor β subunit by PKC is important for regulating the potentiation of GABAA receptor function by neurosteroids.

This work and the studentship to JA, are supported by the MRC

Belelli D et al. (2005). Nature Rev Neurosci 6, 565-575.

McDonald BJ et al. (1998). Nature Neurosci 1, 23-28.

Moss SJ et al. (1996). Int Rev Neurobiol 39, 1-52.