Cell Physiol Biochem 2016;39:1031-1039
DOI: 10.1159/000447810
Published online: August 19, 2016
1031
Fezai et al.: GSK3b Up-Regulates BK Channels
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
© 2016 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Original Paper
Accepted: June 21, 2016
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 Interna-
tional License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution
for commercial purposes as well as any distribution of modied material requires written permission.
DOI: 10.1159/000447810
Published online: August 19, 2016
© 2016 The Author(s)
Published by S. Karger AG, Basel
www.karger.com/cpb
© 2016 The Author(s)
Published by S. Karger AG, Basel
Department of Physiology, Cardiology and Vascular Medicine, University of Tübingen,
Gmelinstr. 5, D-72076 Tübingen, (Germany)
Tel. +49 7071/2972194, Fax +49 7071/295618, E-Mail orian.lang@uni-tuebingen.de
Prof. Dr. Florian Lang
Up-Regulation of the Large-Conductance
Ca
2+
-Activated K
+
Channel by Glycogen
Synthase Kinase GSK3β
Myriam Fezai
a
Musaab Ahmed
a
Zohreh Hosseinzadeh
a,b
Florian Lang
a,c
a
Department of Cardiology, Vascular Medicine and Physiology,
b
Centre for Ophthalmology, Institute
for Ophthalmic Research, University of Tuebingen, Tuebingen,
c
Department of Molecular Medicine II,
Medical Faculty, Heinrich Heine University, Duesseldorf, Germany
Key Words
Large conductance Ca
2+
-activated K
+
channel • Glycogen synthase kinase 3 β • Voltage clamp
• Lithium • Neuronal excitation
Abstract
Background/Aims: The pleotropic functions of the large conductance Ca
2+
-activated K
+
channels (maxi K
+
channel or BK channels) include regulation of neuronal excitation and
cell volume. Kinases participating in those functions include the glycogen synthase kinase
GSK3ß which is under negative control of protein kinase B (PKB/Akt). GSK3ß is inhibited by
the antidepressant Lithium. The present study thus explored whether GSK3ß modies the
activity of BK channels. Methods: cRNA encoding the Ca
2+
insensitive BK channel mutant
BK
M513I+Δ899–903
was injected into Xenopus laevis oocytes without or with additional injection of
cRNA encoding wild-type GSK3ß, inactive
K85R
GSK3ß, or wild-type GSK3ß with wild-type PKB.
K
+
channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel
activity in BK
M513I+Δ899–903
expressing oocytes was signicantly increased by co-expression of
GSK3ß, but not by co-expression of
K85R
GSK3ß. The effect of wild type GSK3ß was abrogated
by additional co-expression of wild-type PKB and by 24 hours incubation with Lithium (1 mM).
Disruption of channel insertion into the cell membrane by brefeldin A (5 µM) was followed by
a decline of the current to a similar extent in oocytes expressing BK and GSK3ß and in oocytes
expressing BK alone. Conclusion: GSK3ß may up-regulate BK channels, an effect disrupted by
Lithium or additional expression of PKB and possibly participating in the regulation of cell volume
and excitability.
Introduction
The large conductance Ca
2+
-activated K
+
channels (maxi K
+
channel or BK channels)
serve a variety of functions including regulation of neuronal excitability [1-23] and cell
volume [24-26].
Cell Physiol Biochem 2016;39:1031-1039
DOI: 10.1159/000447810
Published online: August 19, 2016
1032
Fezai et al.: GSK3b Up-Regulates BK Channels
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
© 2016 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
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
To this end, the Ca
2+
insensitive BK channel mutant BK
was expressed in Xenopus
laevis oocytes without or with additional expression of wild-type GSK3β, inactive mutant
K85R
GSK3ß or wild-type GSK3β together with wild type PKB. The BK channel activity in those
oocytes was determined by dual electrode voltage clamp.
Materials and Methods
Ethical Statement
All experiments conform with the 'European Convention for the Protection of Vertebrate Animals used for
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
Constructs encoding mouse Ca
2+
-insensitive BK channel (BK
) [31, 32] (kindly provided by J
Lingle), wild-type human GSK3ß [33], inactive mutant
K85R
GSK3ß [34], and wild-type PKB [35] were used for
The Ca
2+
-insensitive BK mutant was utilized because
the activity of wild-type BK requires an increase in the intracellular Ca
2+
level in oocytes, which leads to
likely side effects interfering with the measurement [41, 42].
Voltage clamp in Xenopus laevis oocytes
Xenopus laevis
K85R
GSK3ß were injected on the same day
after preparation of the oocytes [37, 45-47]. The oocytes were maintained at 17°C in a solution, containing
(in mM):
2
, 1.8 CaCl
2
5 Sodium pyruvate, supplemented with
Gentamycin (100 mg/l), Tetracycline (50 mg/l mg/l), Theophiline (90
[43, 48]. Lithium Chloride (1 mM or 10 mM) was added where indicated. The voltage clamp experiments
1 s with pulses from -150 to +190 mV in 2 s increments of 20 mV steps from a holding potential of -60 mV.
[35, 49]. The Clampex 9.2 software was used for data acquisition and analysis (Axon Instruments) [35, 47,
50, 51]. (in mM):
2
, 1 MgCl
2,
the bath solution was reached within about 10 s [52, 53].
Statistical analysis
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
p < 0.05 were
Results
activity of the large conductance Ca
2+
-activated K
+
channels (maxi K
+
channel or BK channels).
Ca
2+
-insensitive BK channel (BK
) was injected into
Cell Physiol Biochem 2016;39:1031-1039
DOI: 10.1159/000447810
Published online: August 19, 2016
1033
Fezai et al.: GSK3b Up-Regulates BK Channels
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
© 2016 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Fig. 1. Co-expression of
wild-type GSK3β increases
the K
+
current in BK express-
ing Xenopus laevis oocytes.
A: Representative original
tracings showing currents
in Xenopus oocytes injected
with water (a), expressing
GSK3 alone (b) expressing
BK alone (c) or expressing
BK with additional co-ex-
pression of wild-type GSK3β
(d). The voltage protocol is
shown (not to scale). Cur-
rents were activated by
depolarization from -150
to +190 mV from a holding
potential of -60 mV. B: Arith-
54) of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oo-
cytes injected with water (white circles), expressing GSK3β alone (black triangles) or expressing BK without
(white rectangles) or with (black rectangles) additional co-expression of wild-type GSK3β
curves shown in B between +130 mV and +190 mV in Xenopus oocytes injected with water (dotted bar),
expressing GSK3ß alone (grey bar) or expressing BK without (white bar) or with (black bar) additional
co-expression of wild-type GSK3ß. *** (p
expressing BK alone.
Fig. 2. The effect of GSK3b is
disrupted by the inactivating
mutation
K85R
GSK3β. A: Rep-
resentative original tracings
showing currents in Xenopus
oocytes injected with water
(a), expressing BK alone (b)
or with additional co-expres-
sion of wild-type GSK3ß (c)
or inactive
K85R
GSK3ß
(d). The
voltage protocol is shown
(not to scale). Currents were
activated by depolarization
from -150 to +190 mV from
a holding potential of -60 mV.
a function of the potential dif-
ference across the cell membrane (V) in Xenopus oocytes injected with water (white circles) or expressing
BK without (white rectangles) or with additional co-expression of wild-type GSK3ß (black rectangles), or
inactive
K85R
GSK3β
Xenopus oocytes injected with water (white dotted bar), or expressing BK without ( white bar) or with addi-
tional co-expression of wild-type GSK3ß (black bar) or inactive
K85R
GSK3ß
(grey bar). *** (p<0.001) indicates
Cell Physiol Biochem 2016;39:1031-1039
DOI: 10.1159/000447810
Published online: August 19, 2016
1034
Fezai et al.: GSK3b Up-Regulates BK Channels
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
© 2016 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Xenopus
GSK3ß or, as a negative control, inactive mutant
K85R
GSK3ß. The voltage-gated K
+
current was
determined by dual electrode voltage clamp experiments.
M513I+899–903
into Xenopus
-gated K
+
modify the voltage gated current (Fig. 1).
K85R
gated current in BK
M513I+
expressing Xenopus oocytes (Fig. 2).
(PKB/Akt), wild-type GSK3ß was co-expressed with BK
M513I+
without or with additional
co-expression of PKB. As illustrated in Fig. 3, the co-expression of PKB was followed by
expressing BK
M513I+
together with wild-type GSK3β + wild-type PKB and in oocytes
expressing BK
M513I+
alone.
A further series of experiments explored whether the effect of GSK3β
by the antidepressant Lithium. As illustrated in Fig. 4, a 24 hours Lithium-exposure of
Fig. 3.
Xenopus
oocytes injected with water (white circles) or expressing BK alone (white rectangles), expressing BK with
additional co-expression of wild-type GSK3ß (black rectangles) or expressing BK with additional co-expres-
sion of wild-type GSK3ß and wild-type PKB (black triangles) or expressing BK with additional co-expression
of PKB (white trianlges)-
Xenopus
oocytes injected with water (white dotted bar), or expressing BK without ( white bar) or with additional
co-expression of wild-type GSK3ß (black bar) or with additional expression of both, GSK3ß and PKB
(grey
bar), or with additional co-expression of PKB (grey dotted bar). **(p
-
ference from oocytes expressing BK and GSK3ß.
Cell Physiol Biochem 2016;39:1031-1039
DOI: 10.1159/000447810
Published online: August 19, 2016
1035
Fezai et al.: GSK3b Up-Regulates BK Channels
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
© 2016 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Xenopus oocytes co-expressing BK
M513I+
together with wild-type GSK3β was followed
current in Xenopus oocytes expressing BK
M513I+
alone. A 24 hours of Lithium exposure
M513I+
alone. An 1 hour
+
current in oocytes co-
expressing BK
M513I+
with GSK3β.
In order to test whether the co-expression of wild-type GSK3β stabilizes BK
M513I+
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+
alone and in Xenopus oocytes expressing BK
M513I+
with
additional co-expression of wild-type GSK3β. Thus, GSK3β did not appreciably modify the
channel stability in the cell membrane.
Discussion
The present study uncovers a novel function of glycogen synthase kinase GSK3ß, i.e.
the up-regulation of large conductance Ca
2+
-activated K
+
channels (maxi K
+
channel or BK
channels). Co-expression of the wild-type GSK3ß but not of the inactive mutant
K85R
GSK3ß
Fig. 4.
of the current (I) as a function of the potential difference across the cell membrane (V) in Xenopus oocytes
injected with water (white diamond) or expressing BK alone (white symboles), or expressing BK with ad-
ditional co-expression of wild-type GSK3ß (black symbols) without (rectangles) or with a 24 hours of 1 mM
Lithium exposure (circles) or with 1h/10 mM Lithium exposure (triangle).
B between +130 mV and +190 mV in Xenopus oocytes injected with water (dotted bar), or expressing BK
without (white bar) or with additional co-expression of wild-type GSK3ß (black bar) prior to (left bars) 24
hours of 1mM Lithium exposure (middle bars) or 1 hour of 10mM Lithium exposure (left bar). *** (p<0.001)
p<0.05) indicates statisti-