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. 2000 Sep 15;20(18):6743-51.
doi: 10.1523/JNEUROSCI.20-18-06743.2000.

Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1

D K Meyer  1 , C Olenik, F Hofmann, H Barth, J Leemhuis, I Brünig, K Aktories, W Nörenberg
Affiliations

Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1

D K Meyer et al. J Neurosci. .

Abstract

The role of the cytoskeleton in the activity of GABA(A) receptors was investigated in cultured hippocampal neurons. Receptor currents were measured with the whole-cell patch-clamp technique during repetitive stimulation with 1 microm muscimol. After destruction of the microtubular system with nocodazol, muscimol-induced currents showed a rundown by 78%. A similar rundown was observed when actin fibers were destroyed with latrunculin B or C2 toxin of Clostridium botulinum. Because the small GTPases of the Rho family RhoA, Rac1, and Cdc42 are known to control the organization of actin fibers, we investigated their possible involvement. Inactivation of the GTPases with clostridial toxins, as well as intracellular application of recombinant Rho GTPases, indicated that active Rac1 was necessary for full GABA(A) receptor activity. Immunocytochemical labeling of the receptors showed that the disappearance of receptor clusters in the somatic membrane as induced by muscimol stimulation was enhanced by Rac1 inactivation. It is suggested that Rac1 participates in the regulation of GABA(A) receptor clustering and/or recycling.

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Figures

Fig. 1.
Fig. 1.
Intracellular ATP prevents rundown of GABAA receptor currents in rat hippocampal neurons during repetitive stimulation. Inward currents were evoked by 3 sec pressure application of 1 μm muscimol at 30 sec intervals over 25 min. A, B, Superimposed inward currents after the first (T0) and last (T25) application of muscimol. Currents were recorded at a holding potential of −70 mV with pipette solutions without (A) or with (B) 4 mm Mg-ATP. Thesolid, horizontal linesabove the currenttracesindicate the periods of muscimol pressure application; thedotted lines indicate the zero current level.C, Grouped mean data showing the time course of GABAA receptor current rundown. Currents were expressed as percent of the first muscimol application atT0 (n = 7–8; means ± SEM). The filled squares show the effects of muscimol (1 μm) when applied only twice, i.e., atT0 and T25.
Fig. 2.
Fig. 2.
Microtubules, as well as actin filaments, are involved in the maintenance of GABAA receptor activity in rat hippocampal neurons. A, Time course of GABAA receptor current rundown after repetitive stimulation with muscimol. Pipette solutions contained Mg-ATP (4 mm) alone, Mg-ATP plus C2 toxin (10 ng/ml), Mg-ATP plus latrunculin B (2 μm), and Mg-ATP plus nocodazol (2 μm,open triangles; 10 μm, filled triangles). Parameters for repetitive stimulation with 1 μm muscimol correspond to those of Figure 1. In the experiment with 10 μm nocodazol, the culture was also preincubated with 10 μm nocodazol for at least 4 hr. Currents were expressed as percent of the first muscimol application atT0 (n = 5–6 each; means ± SEM). Immunohistochemistry for β-tubulin III in hippocampal neurons in controls (B), after incubation with nocodazol (10 μm) for 0.5 hr (C) and for 4 hr (D).
Fig. 3.
Fig. 3.
Inactivation of Rho GTPases by clostridial toxins induces a use-dependent GABAA receptor rundown in rat hippocampal neurons. A–C, Superimposed inward currents evoked at T0 andT25. Toxins were added to pipette solutions containing 4 mm Mg-ATP. A, Toxin B (5 ng/ml); B, C3 toxin (6 μg/ml); C, lethal toxin (50 ng/ml). Parameters for repetitive stimulation with 1 μm muscimol correspond to those of Figure 1. Thesolid, horizontal linesabove the currenttracesindicate the periods of muscimol pressure application; thedotted lines indicate the zero current level.D, Grouped mean data showing the time course of GABAA receptor current rundown. Filled squares show effects of muscimol (1 μm) applied only twice, i.e., at T0 andT25, in the presence of intracellular toxin B. Currents were expressed as percent of the first muscimol application at T0 (n = 6–7; means ± SEM).
Fig. 4.
Fig. 4.
Effects of recombinant Rac1 on GABAAreceptor currents in rat hippocampal neurons. Pipette solutions contained Mg-ATP (4 mm) or Mg-ATP plus the recombinant GTPases Rac, its constitutively inactive form Raci together with GTPγS, and Cdc42 together with GTPγS (0.3 mm). Parameters for repetitive stimulation with 1 μm muscimol corresponded to those in Figure 1. Currents were normalized by comparison to the first muscimol application atT0 (n = 5–7; means ± SEM).
Fig. 5.
Fig. 5.
A, Lethal toxin of C. sordellii does not affect the affinity of GABAAreceptors to muscimol. Half-maximal (1 μm), maximal (10 μm), and supramaximal (100 μm) muscimol concentrations were tested in neurons immediately before and after the rundown of GABAA receptor currents (seearrows in inset), which was induced by intracellular lethal toxin (50 ng/ml) plus repetitive stimulation with 1 μm muscimol. Currents were reduced to 32.4 ± 8.5% after 25 min of stimulation. The resulting current amplitudes were normalized by comparison to the maximal response obtained with 100 μm muscimol in the respective individual experiments before induction of rundown. Shown are means ± SEM (n = 5). B, Lethal toxin ofC. sordellii does not affect the reversal potential (Vrev) of GABAAreceptor-mediated currents. In this experiment, rundown of GABAA receptor currents was also induced by intracellular lethal toxin (50 ng/ml) plus repetitive stimulation with 1 μm muscimol. A representative recording of five experiments shows how Vrev of GABAA receptor-mediated currents was assessed (left panel). Fast voltage ramps, 200 msec from the holding potential of −70 to +15 mV, were applied immediately before every application of muscimol (T0 toT25), as well as at approximately each peak response to the agonist (T0 toT25; bottom trace). Also shown are superimposed current traces obtained in response to the voltage-ramps imposed before (I) and during (II) the application of muscimol at the first (T0) and last (T25) challenge with muscimol (left panel, top trace). Under these conditions, the current was decreased to 35.9 ± 10.0% (inset; n = 5). The respective current–voltage (I–V) relationships are shown in the right panel. I–V curves obtained in response to the first (T0) and last (T25) challenge were superimposed. Curves were obtained by subtracting current responses to the voltage ramp in the absence of muscimol from those in its presence (II–I).
Fig. 6.
Fig. 6.
Nocodazol and lethal toxin of C. sordellii enhance the reduction in GABAA receptor clusters induced by repetitive stimulation with muscimol. Immunohistochemistry for α2 subunit of GABAA receptors after destruction of microtubular or actin cytoskeleton with or without subsequent muscimol application. A, Controls; B, muscimol (1 μm for 25 min);C, nocodazol (10 μm for 4 hr);D, lethal toxin (50 ng/ml for 1 hr); E, nocodazol (10 μm for 4 hr) plus subsequent muscimol (1 μm for 25 min); F, lethal toxin (50 ng/ml for 1 hr) plus subsequent muscimol (1 μm for 25 min). Scale bar, 25 μm.
Fig. 7.
Fig. 7.
Insulin prevents the ATP-independent rundown of GABAA receptor currents in rat hippocampal neurons. Inward currents were evoked by 3 sec pressure application of 1 μm muscimol at 30 sec intervals over 25 min. Representative inward currents evoked at the holding potential of −70 mV in amphotericin B perforated patch recording configuration after first (T0) and last (T25) application of muscimol were superimposed and are shown in A–C. A, Control; B, insulin (10 μg/ml) was present in the incubation medium during a 12 hr preincubation period, as well as during the actual experiment; C, protocol (B) was used, and in addition, lethal toxin (50 ng/ml) was added 1 hr before the start of the experiments. The solid, horizontal lines above thecurrent traces indicate the period of muscimol pressure application; the dotted lines indicate the zero current level. The respective time courses of GABAA receptor currents are shown in D. In addition, the effects of muscimol (1 μm) are shown when it was applied only twice, i.e., at T0 andT25, in the absence of extracellular insulin (filled circles). Currents were normalized by comparison to the first muscimol application atT0 (n = 7 each; means ± SEM).
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