SGK3 Sensitivity of Large-Conductance Ca²⁺-Activated K⁺ Channel

The pleotropic effects of the serum & glucocorticoid inducible kinase SGK3 include up-regulation of several transporters [1,2,3,4,5,6,7,8,9] and ion channels [10,11,12,13], including Ca²⁺ channels [10,14] and voltage-gated K⁺ channels [15,16,17,18,19].

At least in theory, SGK3 may similarly modify the large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channels or BK channels, KCNMA1), which are ubiquitously expressed [20,21] and participate in the tuning of a wide variety of functions including regulation of neuronal excitability [22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44], cell volume [45,46,47], vascular tone and blood pressure [48,49], urinary bladder contraction [50], as well as erection [51].

In neurons [20], BK channels contribute to repolarization and fast afterhyperpolarization of action potentials [52,53] and thus influence dendritic Ca²⁺ spikes [54], and neurotransmitter release [55,56,57,58]. They are involved in motor coordination [59], learning and memory [28,60,61,62], circadian rhythm [63,64,65,66], regulation of respiration [67,68,69], hearing [70], and pain [71,72,73,74]. BK channels confer neuroprotection [75,76,77,78]. Defective function of BK channels may lead to epilepsy and paroxysmal dyskinesia [79,80], mental retardation [81,82], autism [83], and schizophrenia [84].

The channels are activated by increase of cytosolic Ca²⁺ activity [20] and are highly sensitive to cell membrane potential [20]. The channel properties are modified by accessory subunits [20,48], BK channel protein abundance and activity could be modified by posttranslational modification including protein phosphorylation, lipidation, glycosylation, and ubiquitination [85,86,87,88,89].

The present study thus explored, whether wild-type SGK3, constitutively active ^S419DSGK3, or catalytically inactive ^K191NSGK3 modify the function 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 SGK3 and/or constitutively active ^S419DSGK3, or catalytically inactive ^K191NSGK3. 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 and 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}) [59,90], wild type SGK3 [91], constitutively active ^S419DSGK3 [92] or inactive ^K191NSGK3 [92] were used for generation of cRNA as described previously [93,94,95,96,97].

Xenopus laevis oocytes were prepared as previously described [98,99]. 20 ng cRNA encoding BK channels and 10 ng of cRNA encoding wild-type, constitutively active or inactive kinase were injected on the same day after preparation of the oocytes [94,100,101,102]. The oocytes were maintained at 17°C in ND96-A, a solution containing (in mM): 88.5 NaCl, 2 KCl, 1 MgC1₂, 1.8 CaC1₂, 2.5 NaOH, 5 HEPES, 5 Sodium pyruvate, Gentamycin (100 mg/l), Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Theophiline (90 mg/l). The solution was titrated to pH 7.4 by addition of NaOH [99,103]. The voltage clamp experiments were performed at room temperature 3 days after the first injection. BK channel currents were elicited by 1 s 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 1 kHz and recorded with a Digidata A/D-D/A converter (1322A Axon Instruments) [104]. The Clampex 9.2 software was used for data acquisition and analysis (Axon Instruments). 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 [105,106].

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 or t-test, as appropriate. Results with p < 0.05 were considered statistically significant.

The present study explored whether the serum & glucocorticoid inducible kinase isoform SGK3 influences the large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channels or BK channels). To this end, cRNA encoding Ca²⁺-insensitive BK channel (BK^{M513I+Δ899-903}) was injected into Xenopus oocytes with or without additional injection of cRNA encoding SGK3 and the voltage-gated K+ current determined by dual electrode voltage clamp experiments.

As shown 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 SGK3 was followed by a modest but significant further increase of the voltage gated current (Fig. 1).

The effect of wild type SGK3 was mimicked by the constitutively active mutant ^S419DSGK3. The additional injection of cRNA encoding ^S419DSGK3 into BK^{M513I+Δ899-903} expressing Xenopus oocytes was again followed by a significant further increase of the voltage gated current (Fig. 2).

In contrast to the injection of cRNA encoding wild-type SGK3 or ^S419DSGK3, the additional injection of cRNA encoding the inactive mutant ^K191NSGK3 did not significantly modify the voltage gated current in BK^{M513I+Δ899-903} expressing Xenopus oocytes (Fig. 3).

A further series of experiments was performed to elucidate whether the effect of SGK3 required Na⁺/K⁺ ATPase activity. To this end, the oocytes were exposed to 1 mM ouabain. As illustrated in Fig. 4, similar to the effects of SGK3 in the absence of ouabain, in the continued presence of ouabain, the additional injection of cRNA encoding SGK3 into BK^{M513I+Δ899-903} expressing Xenopus oocytes was followed by a significant further increase of the voltage gated current (Fig. 4).

The present study uncovers a novel function of the serum & glucocorticoid inducible kinase isoform SGK3, i.e. the up-regulation of large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels). Co-expression of the wild-type SGK3 and of the constitutively active ^S419DSGK3 [92], but not coexpression of the inactive ^K191NSGK3 [92] was followed by a significant increase of the voltage gated current in Xenopus oocytes expressing the Ca²⁺-insensitive BK channel BK^{M513I+Δ899-903}.

The present observations did not attempt to define the cellular mechanisms involved in the up-regulation of BK channel activity by SGK3. In theory, SGK3 could be effective by direct phosphorylation of the channel protein and/or by phosphorylating and thus modifying the activity of other signalling molecules, which in turn influence BK channels. BK channel regulating kinases include AMP activated kinase [107], protein kinase A [108,109], protein kinase C [108,110,111], cGMP-dependent protein kinase (PKG) [112], and cyclin-dependent kinase isoform CDK5 [113]. BK channels are further regulated by WNK4, which downregulates BK channels, an effect requiring ERK and p38 kinase [89]. Interestingly, the effect was abolished by coexpression of SGK1 [89]. SGK3 influences some ion channels by phosphorylating and thus inactivating the ubiquitin ligase Nedd4-2, which otherwise ubiquitinates target proteins thus labelling them for degradation [15,114,115,116,117]. However, whether the large conductance Ca²⁺-activated K⁺ channels are targeted by Nedd4-2, remains uncertain. In theory, SGK3 could have been effective by activation of the Na⁺/K⁺ ATPase [6], which may up-regulate some K⁺ channels [118]. However, Na⁺/K⁺ ATPase inhibitor ouabain did not appreciably blunt the effect of SGK3 on BK.

The functions sensitive to the activity of large conductance Ca²⁺-activated K⁺ channels include regulation of neuronal excitability [26]. Activation of K⁺ channels is expected to hyperpolarize neurons and thus to inhibit excitation. SGK3 contributes to the regulation of neuronal excitability further by regulating glutamate receptors [11,12,119,120] and glutamate transporters [2,3,121]. To which extent the modest upregulation of BK channels contributes to SGK3 sensitive neuronal excitation, remains elusive.

Several K⁺ channels including BK channels are further decisive for regulatory cell volume decrease [122,123,124]. In several cell types cell swelling leads to activation of Ca²⁺ permeable cation channels, increase of cytosolic Ca²⁺ activity, activation of large conductance Ca²⁺-activated K⁺ channels, K⁺ exit, hyperpolarization and potential-driven Cl^- exit [122,123,124]. To which extent SGK3-sensitivity of BK channels contributes to cell volume regulation, remains to be shown, however.

In conclusion, SGK3 elicits a modest stimulating effect on large conductance Ca²⁺-activated K⁺ channels, an effect possibly modifying cell volume regulation and neuroexcitability.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic 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.