Feature Article: Selective modulation of tonically active GABAA receptor functional subgroups by G-proteins and protein kinase C

Abstract

Here we study intracellular mechanisms which regulate inhibitory signaling delivered through continuously (tonically) open ionotropic receptors of γ-aminobutyric acid (GABA) of dentate gyrus granule cells (DGCs). We found that, apart of classical GABA-A receptors (GABA_ARs) which can be activated by GABA binding, a significant part of tonic inhibitory current is delivered by newly discovered spontaneously opening GABA_ARs (s-GABA_ARs), which enter active state without binding of GABA. We have also found that conventional GABA_ARs and s-GABA_ARs are regulated by different intracellular mechanisms, which may overlap and thus induce various signaling repercussions. Our results demonstrate that s-GABA_ARs play a key role in the mechanism that implements DGCs functional role in the brain. On top of that, since regulatory mechanisms under study are affected in a number of pathological states, our results may have broad implications for treatment of neurological disorders.

Keywords: G-proteins; Spontaneously opening GABAA receptors; action potential generation; protein kinase C; tonic inhibitory current.

Figure 1.

Pharmacology of GABA-mediated tonic currents. (a) Example traces of whole-cell continuous recordings from DGCs. From top to bottom: Control, BII added to internal solution, PeTX added to internal solution, PKI added to internal solution. SR-95 (25 μM) abolished spontaneous synaptic activity, whereas Picr (50 μM) induced an outward shift of tonic current. Dashed lines mark time intervals over which I_tonic was averaged for SR-95 (last three minutes before Picr added) and for SR-95+Picr. (b) SR-95 (left columns) and SR-95+Picr (right columns) decrease RMS noise; bars represent change from previous drug application, asterisks indicate significance of difference from zero; n = 5–8, Student’s t-test. Vertical axis signs and bar marks apply to all bar charts. (c) Shifts of I_tonic induced by application of SR-95+Picr, asterisks indicate significance of difference from Control; n = 11–12, Student’s t-test. *P < 0.05, **P < 0.01.

Figure 2.

Modulation of GABA_ARs single-channel parameters by cytoplasmic factors. Traces from top to bottom in a–c: GABA, perfusion solution without GABA_AR ligands, GABA+SR-95, GABA+Picr. Left panel: outside-out patch (OOP). Right panel: nucleated patch (NP) pulled from the same cell. Panel labels given in a and scale bars apply to a–c. (a) Control. (b) Block of PKC by BII (50 nM). (c) Block of G-protein-mediated activity with PeTX (1 μg/mL). (d) Average open time, statistical summary for a–c. (e)Opening frequency, statistical summary for a–c. (f) Open time fraction, statistical summary for a–c. Colour codes apply to d–f. Asterisks denote significance of difference from “Control” NP’s column. *P < 0.05; n = 11–13, Student’s t-test.

Figure 3.

Modulation of GABA_ARs sensitivity to GABA by cytoplasmic factors. a–c: Single-channel openings recorded from nucleated patches. Traces from top to bottom: augmenting concentrations of GABA in perfusion solution. (a) Control. (b) BII (50 nM) in recording pipette. (c) PeTX (1 μg/mL) in recording pipette. Concentrations of GABA given in a and scale bars apply to a–c. (d) GABA_ARs open time per second as a function of GABA concentration. (e)Normalized GABA_ARs open time per second as a function of GABA concentration. Asterisks denote significance of difference between points for PeTX and control. *P < 0.05, **P < 0.01, n = 9–11, Student’s t-test.

Figure 4.

Cytoplasmic factors modulate s-GABA_ARs impact on action potential generation. Minimum current injection which generates APs was used in control; after SR-95 (25 μM) application, the amount of injected current was again adjusted to minimum value generating APs. (a) No ligands added to intracellular solution. (b) BII (50 nM) added to intracellular solution. (c)PeTX (1 μg/mL) added to intracellular solution. Scale bars (bottom) and trace denominations (left) apply to a–c. (d) Bar chart shows “SR-95 to Control” and “Picr to adjusted SR-95” ratios of number of APs (see text for more details). Asterisks display significance of difference of ratios obtained from BII- and PeTX-loaded neurons from ratios generated at the same stage of control experiment. *P < 0.05, n = 6–9, Student’s t-test.