Up-Regulation of the Large-Conductance Ca²⁺-Activated K⁺ Channel by Glycogen Synthase Kinase GSK3β

The large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels) serve a variety of functions including regulation of neuronal excitability [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23] and cell volume [24,25,26].

Kinases implicated in the regulation of neuronal excitability [27] and cell size [28] include the glycogen synthase kinase GSK3β. The kinase is phosphorylated and down-regulated by protein kinase B (PKB/Akt) [29]. GSK3β is inhibited by the antidepressant Lithium [30].

The present study thus explored, whether GSK3β modifies the activity of BK channels. To this end, the Ca²⁺ insensitive BK channel mutant BK^{M513I+Δ899-903} was expressed in Xenopus laevis oocytes without or with additional expression of wild-type GSK3β, inactive mutant ^K85RGSK3ß or wild-type GSK3β together with wild type PKB. The BK channel activity in those oocytes was determined by dual electrode voltage clamp.

All experiments conform with the 'European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes' (Council of Europe No 123, Strasbourg 1985) and were conducted according to the German law for the welfare of animals. The surgical procedures on the adult Xenopus laevis frogs were reviewed and approved by the respective government authority of the state Baden-Württemberg (Regierungspräsidium) prior to the start of the study (Anzeige für Organentnahme nach §36).

Constructs encoding mouse Ca²⁺-insensitive BK channel (BK^{M513I+Δ899-903}) [31,32] (kindly provided by J Lingle), wild-type human GSK3ß [33], inactive mutant ^K85RGSK3ß [34], and wild-type PKB [35] were used for generation of cRNA as described previously [36,37,38,39,40]. The Ca²⁺-insensitive BK mutant was utilized because the activity of wild-type BK requires an increase in the intracellular Ca²⁺ level in oocytes, which leads to likely side effects interfering with the measurement [41,42].

Xenopus laevis oocytes were prepared as previously described [38,43,44]. 20 ng cRNA encoding BK channels and 7.5 ng of cRNA encoding wild-type or inactive kinase ^K85RGSK3ß were injected on the same day after preparation of the oocytes [37,45,46,47]. The oocytes were maintained at 17°C in a solution, containing (in mM): 88.5 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, 2.5 NaOH, 5 HEPES, 5 Sodium pyruvate, supplemented with Gentamycin (100 mg/l), Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Theophiline (90 mg/l) and pH 7.4 [43,48]. Lithium Chloride (1 mM or 10 mM) was added where indicated. The voltage clamp experiments were performed at room temperature 3 days after the first injection. BK channel currents were elicited every 1 s with pulses from -150 to +190 mV in 2 s increments of 20 mV steps from a holding potential of -60 mV. The data were filtered at 2 kHz and recorded with a Digidata A/D-D/A converter (1322A Axon Instruments) [35,49]. The Clampex 9.2 software was used for data acquisition and analysis (Axon Instruments) [35,47,50,51]. The control superfusate (ND96) contained (in mM): 93.5 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 2.5 NaOH and 5 HEPES, pH 7.4. The flow rate of the superfusion was approx. 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [52,53].

Data are provided as means ± SEM, n represents the number of oocytes investigated. As different batches of oocytes may yield different results, comparisons were always made within a given oocyte batch. All voltage clamp experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using ANOVA (Tukey test). Results with p < 0.05 were considered statistically significant.

The present study explored whether glycogen synthase kinase GSK3ß modifies the activity of the large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels). To this end, cRNA encoding Ca²⁺-insensitive BK channel (BK^{M513I+Δ899-903}) was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type GSK3ß or, as a negative control, inactive mutant ^K85RGSK3ß. The voltage-gated K⁺ current was determined by dual electrode voltage clamp experiments.

As illustrated in Fig. 1, the injection of cRNA encoding BK^{M513I+Δ899-903} into Xenopus oocytes was followed by a substantial and significant increase of large voltage-gated K⁺ currents as compared to water-injected oocytes. The additional injection of cRNA encoding wild-type GSK3ß was followed by a moderate but significant further increase of the voltage gated current. The injection of cRNA encoding wild-type GSK3ß alone did not significantly modify the voltage gated current (Fig. 1).

In contrast to the injection of cRNA encoding wild-type GSK3ß, the additional injection of cRNA encoding the inactive mutant ^K85RGSK3ß did not significantly modify the voltage gated current in BK^{M513I+Δ899-903} expressing Xenopus oocytes (Fig. 2).

In order to test whether the effect of GSK3ß could be modified by protein kinase B (PKB/Akt), wild-type GSK3ß was co-expressed with BK^{M513I+Δ899-903}without or with additional co-expression of PKB. As illustrated in Fig. 3, the co-expression of PKB was followed by a significant decline of the voltage gated current which was virtually identical in oocytes expressing BK^{M513I+Δ899-903} together with wild-type GSK3β + wild-type PKB and in oocytes expressing BK^{M513I+Δ899-903} alone.

A further series of experiments explored whether the effect of GSK3β could be modified by the antidepressant Lithium. As illustrated in Fig. 4, a 24 hours Lithium-exposure of Xenopus oocytes co-expressing BK^{M513I+Δ899-903} together with wild-type GSK3β was followed by a significant decline of the voltage gated current to values not significantly higher than the current in Xenopus oocytes expressing BK^{M513I+Δ899-903} alone. A 24 hours of Lithium exposure did not significantly modify the current in oocytes expressing BK^{M513I+Δ899-903} alone. An 1 hour incubation in 10 mM Lithium did not decrease significantly the K⁺ current in oocytes co-expressing BK^{M513I+Δ899-903}with GSK3β.

In order to test whether the co-expression of wild-type GSK3β stabilizes BK^{M513I+Δ899-903} in the cell membrane, the insertion of new channel protein into the cell membrane was prevented by brefeldin A (5 µM). As illustrated in Fig. 5, the brefeldin A treatment was followed by a decay of the voltage gated current. The decay was similar in Xenopus oocytes expressing BK^{M513I+Δ899-903} alone and in Xenopus oocytes expressing BK^{M513I+Δ899-903} with additional co-expression of wild-type GSK3β. Thus, GSK3β did not appreciably modify the channel stability in the cell membrane.

The present study uncovers a novel function of glycogen synthase kinase GSK3ß, i.e. the up-regulation of large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels). Co-expression of the wild-type GSK3ß but not of the inactive mutant ^K85RGSK3ß was followed by a significant increase of the voltage gated current in Xenopus oocytes expressing the Ca²⁺-insensitive BK channel BK^{M513I+Δ899-903}. Expression of GSK3ß alone did not appreciably modify the voltage gated current, indicating that GSK3ß was not effective by modifying an endogenous channel with properties similar to BK^{M513I+Δ899-903}.

The present observations did not define the cellular mechanisms involved in the up-regulation of BK channel activity by GSK3ß. In theory the kinase could be effective by direct phosphorylation of the channel protein or by phosphorylation of proteins involved in the regulation of trafficking or function of the channels. The experiments with brefeldin A suggest that GSK3ß does not affect channel retrieval from the cell membrane.

The effect of GSK3ß was virtually abrogated by the additional co-expression of protein kinase B which is a known negative regulator of GSK3ß activity [29]. Moreover, the effect of GSK3ß was blunted by the antidepressant Lithium, a known inhibitor of GSK3ß [30]. It is tempting to speculate that effects and/or side effects of Lithium may, in part, result from disruption of GSK3ß-induced up-regulation of BK channel activity. However, the present observations do not allow safe conclusions as to the in vivo sensitivity of BK channels to GSK3ß or Lithium.

In theory, the observed effect of GSK3ß on BK channels could contribute to the known effect of the kinase on neuronal excitability [27]. The pleotropic effects of large conductance Ca²⁺-activated K⁺ channels include regulation of neuronal excitability [5]. Activation of K⁺ channels is expected to counteract depolarization and thus excitation. Moreover, the observed GSK3β sensitivity of BK channels could contribute to the impact of the kinase on cell size [28]. K⁺ channels are pivotal molecules in the regulation of cell volume [54,55,56]. Stimulation of BK channel activity is expected to trigger cellular K⁺ loss and secondary Cl^- exit due to hyperpolarization of the cell membrane [54,55,56].

GSK3β up-regulates BK channel activity, an effect possibly modifying cell volume and neuroexcitation.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and Tanja Loch, and technical support by Elfriede Faber. This study was supported by the Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tuebingen University.

The authors of this manuscript state that they do not have any conflict of interests and nothing to disclose.