Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors

Abstract

GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3(S408A,S409A)γ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3(S408A,S409A)δ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4(S443A)β3(S408A,S409A)δ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3(S408,S409) implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by two endogenous neuromodulators.

Keywords: Extrasynaptic GABA(A) receptors; Neurosteroid; Phosphorylation; Protein kinase; Synaptic GABA(A) receptors.

Fig. 1

Staurosporine reduces THDOC–mediated potentiation at α1β3γ2L GABA_A receptors. (A) Mean peak currents recorded from HEK293 cells expressing α1β3γ2L GABA_A receptors in response to EC₂₀ GABA (filled squares) or EC₂₀ GABA + 50 nM THDOC (open squares). Cells either remained untreated (upper panel) or were exposed to 200 nM staurosporine (lower panel). All responses were normalised to the peak current of the first EC₂₀ GABA-activated response, recorded 2 min after achieving the whole-cell recording configuration, designated as t = 0 (100%). (B) Example GABA currents taken at the time points shown as t1–t4 in (A, lower panel) alone, or in the presence of 50 nM THDOC (grey bar), before and after staurosporine treatment (black bar). (C) Bar chart showing the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC in untreated cells (light grey bars, measured at 6 and 26 min, respectively; n = 6), or in treated cells before (black bar) and after (grey bar) 200 nM staurosporine (n = 6). All data points represent mean ± s.e.m. Significant results are indicated by * (P < 0.05, paired t-test).

Fig. 2

PKC activity modulates THDOC–mediated potentiation at α1β3γ2L GABA_A receptors. (A) Mean peak currents recorded from HEK293 cells expressing α1β3γ2L GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells either remained untreated or were treated with 500 nM bisindolylmaleimide-I (BIM-I). Responses were normalised to the first EC₂₀ GABA-activated peak response (t = 0 (100%)). (B) Sample whole-cell currents in the presence and absence of 50 nM THDOC (grey bar) before and after BIM-I treatment (black bar). Representative currents are shown from the time points (t) indicated in (A: lower panel). (C) Bar chart for the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC in untreated cells (light grey bars, measured at 6 and 52 min, respectively; n = 6), or in treated cells before (black bar) and after (grey bar) 500 nM BIM-I treatment (n = 5). *P < 0.05, paired t-test.

Fig. 3

PKC but not PKA increases THDOC–mediated potentiation of α1β3γ2L GABA_A receptor currents. (A & B), Top panels: mean peak currents recorded for α1β3γ2L GABA_A receptors expressed in HEK cells in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were untreated or were treated with 100 nM PMA (A) or 300 μM cAMP (B). All currents are normalised as in Fig. 1. Middle panels: typical GABA currents showing potentiation by 50 nM THDOC (grey bar) before and after PMA (A) or cAMP (B). Currents are taken at the respective time points (t1-t4, t5-t8). Lower panels: bar charts for the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC in untreated cells (light grey bars, n = 4–6) or in treated cells before (black bars) and after (grey bars) 100 nM PMA (A; n = 6) or 300 μM cAMP (B; n = 4) treatment. *P < 0.05, paired t-test.

Fig. 4

Effect of PMA on the potentiation of α1β3γ2L GABA_A receptor currents by varying concentrations of THDOC. Bar chart for the mean potentiations of EC₂₀ GABA-activated responses elicited by THDOC (0.1–100 nM) before (black bars) and after (grey bars) 100 nM PMA (n = 5–11). All data points represent mean ± s.e.m. *P < 0.05, paired t-test.

Fig. 5

Residues β3^S408A,S409A mediate the staurosporine–induced reduction in THDOC potentiation at α1β3γ2L GABA_A receptors. (A) Mean peak GABA currents for α1β3^S408Aγ2L (top panel), α1β3^S409Aγ2L (middle panel) and α1β3^S408A,S409Aγ2L (lower panel; all n = 5) GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were treated with 200 nM staurosporine. All responses were normalised as in Fig. 1. (B) whole-cell currents showing potentiation by 50 nM THDOC (grey bars) before and after staurosporine treatment (black bar) at α1β3^S408A,S409Aγ2L GABA_A receptors. (C) Bar chart of the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC before (black bars) and after (grey bars) staurosporine treatment for α1β3γ2L (n = 6), α1β3^S408Aγ2L (n = 5), α1β3^S409Aγ2L (n = 5) and α1β3^S408A,S409Aγ2L (n = 5) GABA_A receptors. Data for α1β3γ2L is taken from Fig. 1. *P < 0.05, paired t-test.

Fig. 6

Modulation of PKC activity does not alter neurosteroid potentiation at α1β3^S408A,S409Aγ2L GABA_A receptors. (A & C) Mean peak GABA currents for α1β3^S408A,S409Aγ2L (n = 4) GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were treated with either 100 nM PMA (A) or 500 nM Bisindolylmaleimide I (BIM-I; C). (B & D) Bar charts for the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC before (black bars) and after (grey bars): PMA (B) in cells expressing α1β3γ2L (n = 6), α1β3^S408Aγ2L (n = 4), α1β3^S409Aγ2L (n = 4) or α1β3^S408A,S409Aγ2L (n = 5); or BIM-I (D) in cells expressing α1β3γ2L (n = 5) or α1β3^S408A,S409Aγ2L (n = 4) GABA_A receptors. Data for α1β3γ2L is taken from Fig. 2, Fig. 3A. *P < 0.05, paired t-test.

Fig. 7

THDOC enhances phosphorylation of β3^S408,S409 by binding to the GABA_A receptor. (A,C) Representative Western blots showing the level of β3 subunit phosphorylation in HEK293 cells expressing (A) α1β3γ2L or (C) α1^Q241Wβ3γ2L GABA_A receptors. Cells were either untreated (UT) or exposed to 50 nM THDOC for 5, 10 or 20 min or 100 nM PMA for 30 min (as indicated) and probed with β3 or phospho-β3 anti-sera. Cells transfected with α1β3^S408A,S409Aγ2L or α1γ2L GABA_A receptor subunits were used as controls. (B, D) Bar charts showing average levels of phosphorylated β3 subunits in cells expressing α1β3γ2L (B: black bars; n = 3) or α1^Q241Wβ3γ2L (D: grey bars; n = 3), in UT cells or after 50 nM THDOC for 5, 10 or 20 min, or 100 nM PMA (D) for 30 min. *P < 0.05, ANOVA with Dunnett's post hoc test.

Fig. 8

Staurosporine reduces THDOC potentiation at α4β3δ GABA_A receptors. (A) Mean peak currents for α4β3δ GABA_A receptors activated by EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were either untreated (upper panel) or exposed to 200 nM staurosporine (lower panel). (B) Sample whole-cell currents in the absence and presence of 50 nM THDOC (grey bar) before and after 200 nM staurosporine treatment (black bar). (C) Bar chart showing the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC in untreated cells (light grey bars, measured at 6 and 26 min, respectively; n = 5) or in treated cells before (black bar) and after (grey bar) 200 nM staurosporine treatment (n = 9). Responses were normalised to the peak current recorded at 2 min. *P < 0.05, paired t-test.

Fig. 9

Staurosporine reduces THDOC potentiation at α4β3^S408A,S409Aδ GABA_A receptors. (A) Mean peak currents for α4β3^S408A,S409Aδ GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were treated with 200 nM staurosporine. (B) Bar chart showing the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC before (black bars) and after (grey bars) staurosporine treatment in cells expressing α4β3δ (n = 9) or α4β3^S408A,S409Aδ (n = 7) GABA_A receptors. Data for α4β3δ receptors is taken from Fig. 8. *P < 0.05, paired t-test.

Fig. 10

Staurosporine reduces THDOC potentiation at α4^S443Aβ3δ, but not at α4^S443Aβ3^S408A,S409Aδ GABA_A receptors. (A) Mean peak currents recorded from HEK293 cells expressing α4^S443Aβ3δ (top panel: n = 7) or α4^S443Aβ3^S408A,S409Aδ (bottom panel: n = 7) GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were treated with 200 nM staurosporine. (B) Bar chart showing the mean potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC before (black bars) and after (grey bars) staurosporine treatment in cells expressing α4β3δ (n = 9), α4^S443Aβ3δ (n = 7) or α4^S443Aβ3^S408A,S409Aδ (n = 7) GABA_A receptors. Data for α4β3δ receptors is taken from Fig. 8. *P < 0.05, paired t-test.

Supplementary Fig. 1

Inhibition of PKA or PKG does not affect THDOC–mediated potentiation at α1β3γ2L GABA_A receptors. (A & C) Mean peak currents recorded for α1β3γ2L GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were either untreated or exposed to 500 nM myristoylated PKA inhibitor peptide 14-22 amide (PKAI; A) or 3 μM KT5823 (C). (B & D) Bar charts showing the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC in untreated cells (light grey bars; n = 6) or in treated cells before (black bars) and after (grey bars) 500 nM PKAI (B; n = 5) or 3 μM KT5823 (D; n = 6) treatment. *P < 0.05, paired t-test.

Supplementary Fig. 2

Staurosporine reduces THDOC potentiation at α1β3γ2L^S327A,S343A GABA_A receptors. (A) Mean peak currents recorded from HEK293 cells expressing α1β3γ2L^S327A,S343A (n = 4) GABA_A receptors in response to EC₂₀ GABA or EC₂₀ GABA + 50 nM THDOC. Cells were treated with 200 nM staurosporine. (B) Bar chart showing the potentiation of EC₂₀ GABA-activated currents by 50 nM THDOC before (black bars) and after (grey bars) staurosporine for α1β3γ2L (n = 6) or α1β3γ2L^S327A,S343A (n = 4) GABA_A receptors. Data for α1β3γ2L GABA_A receptors is taken from Fig. 1. *P < 0.05, paired t-test.