Background: The voltage gated K+ channels Kv1.3 and Kv1.5 contribute to the orchestration of cell proliferation. Kinases participating in the regulation of cell proliferation include protein kinase B (PKB/Akt). The present study thus explored whether PKB/Akt modifies the abundance and function of Kv1.3 and Kv1.5. Methods: Kv1.3 or Kv1.5 was expressed in Xenopus laevis oocytes with or without wild-type PKB/Akt, constitutively active T308D/S473DPKB/Akt or inactive T308A/S473APKB/Akt. The channel activity was quantified utilizing dual electrode voltage clamp. Moreover, HA-tagged Kv1.5 protein was determined utilizing chemiluminescence. Results: Voltage gated K+ currents were observed in Kv1.3 or Kv1.5 expressing oocytes but not in water-injected oocytes or in oocytes expressing PKB/Akt alone. Co-expression of PKB/Akt or T308D/S473DPKB/Akt, but not co-expression of T308A/S473APKB/Akt significantly increased the voltage gated current in both Kv1.3 and Kv1.5 expressing oocytes. As shown for Kv1.5, co-expression of PKB/Akt enhanced the channel protein abundance in the cell membrane. In Kv1.5 expressing oocytes voltage gated current decreased following inhibition of carrier insertion by brefeldin A (5 µM) to similarly low values in the absence and presence of PKB/Akt, suggesting that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates both, Kv1.3 and Kv1.5 K+ channels.

Keywords:
Voltage gated K+ channels, Protein kinase B, PKB/Akt, Chemiluminescence, Brefeldin

The pleotropic functions of protein kinase B (PKB/Akt) include the regulation of cell survival and cell proliferation [1,2,3,4,5,6,7,8,9]. Cell proliferation is further tuned by regulation of ion channels [10,11,12,13,14]. As a matter of fact, PKB/Akt has previously been shown to stimulate several ion channels, such Ca2+ channels [15,16], CFTR Cl- channels [17], inwardly rectifying K+ channels [18] as well as delayed rectifying hERG [19] and KCNQ1 [20] K+ channels.

The present study explored, whether PKB/Akt regulates the voltage gated K+ channels Kv1.3/Kv1.5, which are both implicated in the regulation of cell proliferation [21,22,23,24,25,26]. Agonists contributing to the regulation Kv1.3 or Kv1.5 channels include insulin [27,28,29] and growth factors such as insulin like growth factor IGF1 [30] epidermal growth factor [31] and brain-derived neurotrophic factor (BDNF) [27,32,33]. Mechanisms mediating insulin and growth factor dependent regulation of Kv1.3 and Kv1.5 include tyrosine kinases [34,35] and PI3 kinase signalling with activation of the serum & glucocorticoid inducible kinase and PKB/Akt isoforms [30,36].

In order to analyse the effect of PKB/Akt on Kv1.3/Kv1.5 channels, voltage gated current was estimated utilizing the dual-electrode voltage-clamp in Xenopus laevis oocytes expressing Kv1.3 or Kv1.5 alone or together with wild-type PKB/Akt, constitutively active T308D/S473DPKB/Akt or inactive T308A/S473APKB/Akt.

As a result, co-expression of PKB/Akt or of T308D/S473DPKB/Akt, but not of T308A/S473APKB/Akt enhanced voltage gated current in Kv1.3 or Kv1.5 expressing Xenopus oocytes, an effect at least partially due to enhanced insertion of channel protein into the cell membrane.

Constructs

The cDNA constructs encoding wild-type PKB/Akt [17], constitutively active T308D/S473DPKB/Akt and inactive T308A/S473APKB/Akt [37], wild-type Kv1.3 (mouse) [38], and/or wild type Kv1.5 or/and Kv1.5-HA (mouse) [39] were used for generation of cRNA as described previously [40,41]

Electrophysiological recordings in Xenopus oocytes

Xenopus laevis oocytes were prepared as previously described [42]. cRNA encoding PKB/Akt, T308D/S473DPKB/Akt or T308A/S473APKB/Akt (10 ng) and cRNA encoding Kv1.3 or Kv1.5 (2.5 ng) was injected on the same day after the preparation of the Xenopus oocytes. All experiments were performed at room temperature (about 22° C) 3 days after the injection [19]. Two-electrode voltage-clamp recordings were performed at a holding potential of -100 mV. The currents were recorded following 2 second depolarizing pulses ranging from −80 to +50 mV in 10 mV and 20 s increments for Kv1.5 or 15 s increments for Kv1.3. The data were filtered at 2 kHz and recorded with a Digidata 1322A A/D-D/A converter and Clampex.9.2 software for data acquisition (Axon Instruments). The analysis of the data was performed with Clampfit 9.2 (Axon Instruments) software [43,44]. The oocytes were maintained at 17°C in ND96 solution containing: 88.5 mM NaCl, 2 mM KCl, 1 mM MgC12, 1.8 mM CaC12, 5 mM HEPES; Tretracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Refobacin (100 mg/l) and Theophylin (90 mg/l) as well as sodium pyruvate (5 mM) were added to the ND96; pH was adjusted to 7.4 by addition of NaOH. The control superfusate (ND96) contained 93.5 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH was adjusted to 7.4 by addition of NaOH (2.5 mM) [45]. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [46,47].

Detection of Kv1.5 cell surface expression by chemiluminescence

To determine HA-Kv1.5 cell surface expression by chemiluminescence [48], oocytes were incubated with mouse monoclonal anti-HA antibody conjugated to horseradish peroxidase (1:1000, Miltenyi Biotec Inc, CA, USA). Individual oocytes were placed in 96 well plates with 20 µl of SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA) and chemiluminescence of single oocytes was quantified in a luminometer (Walter Wallac 2 plate reader, Perkin Elmer, Juegesheim, Germany) by integrating the signal over a period of 1 sec. Results display normalized relative light units.

Statistical analysis

Data are provided as arithmetic means ± SEM; n represents the number of oocytes investigated. All oocyte experiments were repeated with at least three batches of oocytes and in all repetitions qualitatively similar data were obtained. All data were tested for significance by using ANOVA (Tukey or Kruskal-Wallis test) or t-test, as appropriate. Results with p<0.05 were considered statistically significant.

In order to define the impact of protein kinase B (PKB/Akt) on the voltage gated K+ channels Kv1.3 and Kv1.5, cRNA encoding the channels was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding PKB/Akt. Voltage gated K+ channel current was then determined with the dual-electrode voltage-clamp technique. As illustrated in Fig. 1, no appreciable currents were observed in water-injected Xenopus oocytes, indicating that expression of endogenous voltage gated K+ channels was minimal. Injection of cRNA encoding Kv1.3 was, however, followed by the appearance of robust voltage gated current (Fig. 1). Co-expression of wild type PKB/Akt was followed by a marked increase of the voltage gated current by 17 ± 2% (Fig. 1).

Fig. 1
Fig. 1. Co-expression of wild type PKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b) or expressing Kv1.3 with additional co-expression of wild-type PKB/Akt (c). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 30) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (white circles) or expressing Kv1.3 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 15 - 30) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. **(p<0.01) indicates statistically significant difference from oocytes expressing Kv1.3 alone.
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Co-expression of wild type PKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b) or expressing Kv1.3 with additional co-expression of wild-type PKB/Akt (c). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 30) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (white circles) or expressing Kv1.3 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 15 - 30) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. **(p<0.01) indicates statistically significant difference from oocytes expressing Kv1.3 alone.

Fig. 1
Fig. 1. Co-expression of wild type PKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b) or expressing Kv1.3 with additional co-expression of wild-type PKB/Akt (c). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 30) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (white circles) or expressing Kv1.3 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 15 - 30) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. **(p<0.01) indicates statistically significant difference from oocytes expressing Kv1.3 alone.
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Co-expression of wild type PKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b) or expressing Kv1.3 with additional co-expression of wild-type PKB/Akt (c). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 30) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (white circles) or expressing Kv1.3 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 15 - 30) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. **(p<0.01) indicates statistically significant difference from oocytes expressing Kv1.3 alone.

Close modal

The effect of wild type PKB/Akt was mimicked by the constitutively active T308D/S473DPKB/Akt mutant, which increased the current by 17 ± 2%. In contrast, the inactive mutant T308A/S473APKB/Akt did not significantly modify the voltage gated current in Kv1.3 expressing Xenopus oocytes (Fig. 2).

Fig. 2
Fig. 2. Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b), expressing Kv1.3 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.3 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 12 - 19) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.3 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 12 - 19) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308AS/473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.3 alone.
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Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b), expressing Kv1.3 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.3 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 12 - 19) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.3 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 12 - 19) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308AS/473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.3 alone.

Fig. 2
Fig. 2. Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b), expressing Kv1.3 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.3 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 12 - 19) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.3 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 12 - 19) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308AS/473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.3 alone.
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Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.3-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.3 alone (b), expressing Kv1.3 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.3 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 15 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 12 - 19) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.3 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 12 - 19) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.3 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308AS/473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.3 alone.

Close modal

Similar observations were made in Xenopus oocytes expressing Kv1.5. Injection of cRNA encoding Kv1.5 was again followed by the appearance of robust voltage gated currents (Fig. 3), which were again markedly increased (by 11 ± 1%) following co-expression of wild type PKB/Akt (Fig. 3). The expression of PKB/Akt alone, however, failed to generate a voltage gated current.

Fig. 3
Fig. 3. Co-expression of wild type PKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing PKB/Akt alone (b), expressing Kv1.5 alone (c) or expressing Kv1.5 with additional co-expression of wild-type PKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 23 - 47) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles), or expressing PKB/Akt alone (white circles) or expressing Kv1.5 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 23 - 47) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing PKB/Akt alone (striped bar), or expressing Kv1.5 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.
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Co-expression of wild type PKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing PKB/Akt alone (b), expressing Kv1.5 alone (c) or expressing Kv1.5 with additional co-expression of wild-type PKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 23 - 47) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles), or expressing PKB/Akt alone (white circles) or expressing Kv1.5 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 23 - 47) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing PKB/Akt alone (striped bar), or expressing Kv1.5 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.

Fig. 3
Fig. 3. Co-expression of wild type PKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing PKB/Akt alone (b), expressing Kv1.5 alone (c) or expressing Kv1.5 with additional co-expression of wild-type PKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 23 - 47) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles), or expressing PKB/Akt alone (white circles) or expressing Kv1.5 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 23 - 47) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing PKB/Akt alone (striped bar), or expressing Kv1.5 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.
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Co-expression of wild type PKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing PKB/Akt alone (b), expressing Kv1.5 alone (c) or expressing Kv1.5 with additional co-expression of wild-type PKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 23 - 47) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles), or expressing PKB/Akt alone (white circles) or expressing Kv1.5 without (white squares) or with (white triangles) additional co-expression of wild-type PKB/Akt. C: Arithmetic means ± SEM (n = 23 - 47) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing PKB/Akt alone (striped bar), or expressing Kv1.5 without (white bar) or with (black bar) additional co-expression of wild-type PKB/Akt. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.

Close modal

As illustrated in Fig. 4, the effect of wild type PKB/Akt was mimicked by the constitutively active T308D/S473DPKB/Akt mutant, which increased the current by 27 ± 1%. In contrast, the current was not significantly modified by the inactive mutant T308A/S473APKB/Akt (Fig. 4).

Fig. 4
Fig. 4. Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.5 alone (b) or expressing Kv1.5 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.5 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 25) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.5 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 15 - 25) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.5 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308A/S473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.
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Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.5 alone (b) or expressing Kv1.5 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.5 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 25) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.5 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 15 - 25) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.5 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308A/S473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.

Fig. 4
Fig. 4. Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.5 alone (b) or expressing Kv1.5 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.5 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 25) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.5 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 15 - 25) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.5 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308A/S473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.
View largeDownload slide

Co-expression of constitutively active T308D/S473DPKB/Akt but not of inactive T308A/S473APKB/Akt increased the K+ current in Kv1.5-expressing Xenopus laevis oocytes. A: Representative original tracings showing currents in Xenopus oocytes injected with water (a), expressing Kv1.5 alone (b) or expressing Kv1.5 with additional co-expression of constitutively active T308D/S473DPKB/Akt (c) or expressing Kv1.5 with additional co-expression of inactive T308A/S473APKB/Akt (d). The voltage protocol is shown (not to scale). Currents were activated by depolarization from -80 to +50 mV in 20 second increments of 10 mV steps from a holding potential of -100 mV. B: Arithmetic means ± SEM (n = 15 - 25) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes injected with water (black circles) or expressing Kv1.5 without (white squares) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (white triangles) or of inactive T308A/S473APKB/Akt (white circles). C: Arithmetic means ± SEM (n = 15 - 25) of the conductance calculated by linear fit of I/V-curves shown in B between 20 mV and 50 mV in Xenopus oocytes injected with water (dotted bar), or expressing Kv1.5 without (white bar) or with additional co-expression of constitutively active T308D/S473DPKB/Akt (black bar) or of inactive T308A/S473APKB/Akt (grey bar). * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5 alone.

Close modal

The enhanced channel activity could have resulted from increased channel protein abundance in the plasma membrane. To explore this possibility, chemiluminescence experiments were performed. As illustrated in Fig. 5, the co-expression of PKB/Akt resulted in a significant increase (by 32 ± 10%) of the Kv1.5-HA channel protein abundance within the oocyte membrane.

Fig. 5
Fig. 5. PKB/Akt increased Kv1.5-HA protein abundance within the oocyte membrane. Arithmetic means ± SEM (n = 94 - 98) of normalized Kv1.5-HA chemiluminescence in Xenopus oocytes injected with water (dotted bar), expressing Kv1.5-HA alone (white bar) or expressing Kv1.5-HA together with wild-type PKB/Akt (black bar). For normalization, the chemiluminescence was divided by the chemiluminescence of oocytes expressing Kv1.5-HA alone. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5-HA alone.
View largeDownload slide

PKB/Akt increased Kv1.5-HA protein abundance within the oocyte membrane. Arithmetic means ± SEM (n = 94 - 98) of normalized Kv1.5-HA chemiluminescence in Xenopus oocytes injected with water (dotted bar), expressing Kv1.5-HA alone (white bar) or expressing Kv1.5-HA together with wild-type PKB/Akt (black bar). For normalization, the chemiluminescence was divided by the chemiluminescence of oocytes expressing Kv1.5-HA alone. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5-HA alone.

Fig. 5
Fig. 5. PKB/Akt increased Kv1.5-HA protein abundance within the oocyte membrane. Arithmetic means ± SEM (n = 94 - 98) of normalized Kv1.5-HA chemiluminescence in Xenopus oocytes injected with water (dotted bar), expressing Kv1.5-HA alone (white bar) or expressing Kv1.5-HA together with wild-type PKB/Akt (black bar). For normalization, the chemiluminescence was divided by the chemiluminescence of oocytes expressing Kv1.5-HA alone. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5-HA alone.
View largeDownload slide

PKB/Akt increased Kv1.5-HA protein abundance within the oocyte membrane. Arithmetic means ± SEM (n = 94 - 98) of normalized Kv1.5-HA chemiluminescence in Xenopus oocytes injected with water (dotted bar), expressing Kv1.5-HA alone (white bar) or expressing Kv1.5-HA together with wild-type PKB/Akt (black bar). For normalization, the chemiluminescence was divided by the chemiluminescence of oocytes expressing Kv1.5-HA alone. * (p<0.05) indicates statistically significant difference from oocytes expressing Kv1.5-HA alone.

Close modal

In theory, PKB/Akt could up-regulate Kv1.5 by fostering insertion of channel protein into the cell membrane or by delaying retrieval of channel protein from the cell membrane. To discriminate between these two possibilities, Kv1.5-expressing Xenopus oocytes were treated with 5 µM brefeldin A, an inhibitor of protein insertion into the cell membrane. As shown in Fig. 6, brefeldin A decreased the voltage gated current to similar low values in oocytes expressing Kv1.5 alone and in oocytes expressing Kv1.5 together with PKB/Akt. Apparently, retrieval of carrier protein from the cell membrane was not significantly different between Xenopus oocytes expressing Kv1.5 alone and Xenopus oocytes expressing Kv1.5 together with PKB/Akt. Therefore, PKB/Akt up-regulated Kv1.5 channels most likely by stimulating channel insertion into the cell membrane.

Fig. 6
Fig. 6. Effect of brefeldin A on Xenopus laevis oocytes expressing either Kv1.5 alone or with PKB/Akt. Arithmetic means ± SEM (n = 16 - 19) of the conductance calculated by respective linear fit of I/V-curves between 20 mV and 50 mV in Xenopus oocytes injected with cRNA encoding Kv1.5 without (white bars) or with (black bars) PKB/Akt and incubated without (left bars) or with 5 µM brefeldin A for 16 hours (middle bars) or 24 hours (right bars) prior to the measurement. **(p<0.01) indicates significant difference from the absence of PKB/Akt. ### (p<0.001) indicates significant difference from the absence of brefeldin A.
View largeDownload slide

Effect of brefeldin A on Xenopus laevis oocytes expressing either Kv1.5 alone or with PKB/Akt. Arithmetic means ± SEM (n = 16 - 19) of the conductance calculated by respective linear fit of I/V-curves between 20 mV and 50 mV in Xenopus oocytes injected with cRNA encoding Kv1.5 without (white bars) or with (black bars) PKB/Akt and incubated without (left bars) or with 5 µM brefeldin A for 16 hours (middle bars) or 24 hours (right bars) prior to the measurement. **(p<0.01) indicates significant difference from the absence of PKB/Akt. ### (p<0.001) indicates significant difference from the absence of brefeldin A.

Fig. 6
Fig. 6. Effect of brefeldin A on Xenopus laevis oocytes expressing either Kv1.5 alone or with PKB/Akt. Arithmetic means ± SEM (n = 16 - 19) of the conductance calculated by respective linear fit of I/V-curves between 20 mV and 50 mV in Xenopus oocytes injected with cRNA encoding Kv1.5 without (white bars) or with (black bars) PKB/Akt and incubated without (left bars) or with 5 µM brefeldin A for 16 hours (middle bars) or 24 hours (right bars) prior to the measurement. **(p<0.01) indicates significant difference from the absence of PKB/Akt. ### (p<0.001) indicates significant difference from the absence of brefeldin A.
View largeDownload slide

Effect of brefeldin A on Xenopus laevis oocytes expressing either Kv1.5 alone or with PKB/Akt. Arithmetic means ± SEM (n = 16 - 19) of the conductance calculated by respective linear fit of I/V-curves between 20 mV and 50 mV in Xenopus oocytes injected with cRNA encoding Kv1.5 without (white bars) or with (black bars) PKB/Akt and incubated without (left bars) or with 5 µM brefeldin A for 16 hours (middle bars) or 24 hours (right bars) prior to the measurement. **(p<0.01) indicates significant difference from the absence of PKB/Akt. ### (p<0.001) indicates significant difference from the absence of brefeldin A.

Close modal

The present study reveals a novel function of PKB/Akt, i.e. the up-regulation of the voltage gated K+ channels Kv1.3 and Kv1.5. The effect was mimicked by the constitutively active T308D/S473DPKB/Akt, but not by the inactive mutant T308A/S473APKB/Akt. The experiments utilizing brefeldin A suggest that PKB/Akt stimulates insertion of channel protein into the cell membrane rather than delaying retrieval of channel protein from the cell membrane.

The regulation of voltage gated K+ channels could, at least in theory, impact on cell volume [49]. K+ exit through K+ channels generates a cell-negative potential difference across the cell membrane driving Cl- exit. The up-regulation of K+ channels is expected to hyperpolarize the cell membrane thus driving Cl- exit. PKB/Akt activation has, however, been shown to increase cell size [50] and T-cells overexpressing active Akt1 are enlarged [51]. The swelling effect of PKB/Akt may result from cellular ion accumulation by the Na+/H+ exchanger [52].

Up-regulation of Kv1.3 or Kv1.5 K+ channels may further affect cell proliferation, which, at least in some cell types, requires activity of Kv1.3 channels [21,22,23,24,25] and/or Kv1.5 channels [22,26]. Accordingly, up-regulation of Kv1.3 and/or Kv1.5 may participate in the stimulation of cell proliferation and tumor-formation by PKB/Akt [1,2,3,4,5,6,7,8,9].

Kv1.3 and Kv1.5 channels serve a number of further functions including Kv1.3 sensitive immune response & inflammation [23,53,54,55] or diabetes [56], as well as Kv1.5 sensitive cardiac atrial repolarization [57,58,59,60], inhibition of insulin release [61], and regulation of pulmonary artery smooth muscle cell activity [62,63].

In conclusion, PKB/Akt up-regulates the voltage gated K+ channels Kv1.3 and Kv1.5, an effect possibly participating in the orchestration of cell proliferation and further Kv1.3 and Kv1.5 sensitive functions.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and Tanja Loch and the technical support by Elfriede Faber. This study was supported by the Deutsche Forschungsgemeinschaft, GRK 1302, SFB 773 B4/A1, La 315/13-3, and Open Access Publishing Fund of Tuebingen University.

The authors declare that they have nothing to disclose.

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J. Warsi and M. Fezai contributed equally thus share first authorship.

© 2015 The Author(s) Published by S. Karger AG, Basel
2015
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