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The FASEB Journal article fj.12-208132. Published online July 5, 2012.
The FASEB Journal • Research Communication
AMP-activated protein kinase in BK-channel regulation
and protection against hearing loss following
acoustic overstimulation
Michael Föller,*,§,1 Mirko Jaumann,†,1 Juliane Dettling,† Ambrish Saxena,*
Tatsiana Pakladok,* Carlos Munoz,* Peter Ruth,‡ Mentor Sopjani,*,储
Guiscard Seebohm,¶ Lukas Rüttiger,† Marlies Knipper,†,2 and Florian Lang*,2,3
*Department of Physiology,†Department of Otolaryngology, Tübingen Hearing Research Centre,
Molecular Physiology of Hearing, and ‡Institute of Pharmacy, Department of Pharmacology and
Toxicology, University of Tübingen, Tübingen, Germany; §Campbell Family Institute for Breast
Cancer Research, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada;
储
Faculty of Medicine, University of Pristina, Pristina, Kosovo; and ¶Institut für Genetik von
Herzerkrankungen, Universitätsklinik Münster, Münster, Germany
ABSTRACT
The energy-sensing AMP-activated serine/
threonine protein kinase (AMPK) confers cell survival in
part by stimulation of cellular energy production and
limitation of cellular energy utilization. AMPK-sensitive
functions further include activities of epithelial Naⴙ channel ENaC and voltage-gated Kⴙ channel KCNE1/
KCNQ1. AMPK is activated by an increased cytosolic
Ca2ⴙ concentration. The present study explored whether
AMPK regulates the Ca2ⴙ-sensitive large conductance and
voltage-gated potassium (BK) channel. cRNA encoding
BK channel was injected into Xenopus oocytes with
and without additional injection of wild-type AMPK
(AMPK␣1ⴙAMPK␤1ⴙAMPK␥1), constitutively active
AMPK␥R70Q, or inactive AMPK␣K45R. BK-channel activity
was determined utilizing the 2-electrode voltage-clamp.
Moreover, BK-channel protein abundance in the cell
membrane was determined by confocal immunomicroscopy. As BK channels are expressed in outer hair cells
(OHC) of the inner ear and lack of BK channels increases
noise vulnerability, OHC BK-channel expression was examined by immunohistochemistry and hearing function
analyzed by auditory brain stem response measurements
in AMPK␣1-deficient mice (ampkⴚ/ⴚ) and in wild-type
mice (ampkⴙ/ⴙ). As a result, coexpression of AMPK or
AMPK␥R70Q but not of AMPK␣K45R significantly enhanced
BK-channel-mediated currents and BK-channel protein
abundance in the oocyte cell membrane. BK-channel
expression in the inner ear was lower in ampkⴚ/ⴚmice
than in ampkⴙ/ⴙ mice. The hearing thresholds prior to
and immediately after an acoustic overexposure were
similar in ampkⴚ/ⴚ and ampkⴙ/ⴙ mice. However, the
recovery from the acoustic trauma was significantly impaired in ampkⴚ/ⴚmice compared to ampkⴙ/ⴙ mice. In
summary, AMPK is a potent regulator of BK channels. It
may thus participate in the signaling cascades that protect
the inner ear from damage following acoustic overstimulation.—Föller, M., Jaumann, M., Dettling, J., Saxena, A.,
Pakladok, T., Munoz, C., Ruth, P., Sopjani, M., Seebohm,
G., Rüttiger, L., Knipper, M., Lang, F. AMP-activated
protein kinase in BK-channel regulation and protection
against hearing loss following acoustic overstimulation.
FASEB J. 26, 000 – 000 (2012). www.fasebj.org
Key Words: energy depletion 䡠 Ca2⫹ activated K⫹ channels
䡠 inner ear 䡠 acoustic trauma
The AMP-activated protein kinase (AMPK) senses the
cytosolic AMP/ATP concentration ratio and thus the
energy status of the cell (1, 2). To replenish cellular ATP
levels (3), AMPK enhances glucose uptake, glycolysis, fatty
acid oxidation, and the activity of enzymes required for
ATP production (2, 4 –27). AMPK further enhances
phagocytosis (28) and autophagy (29). However, AMPK
decreases energy consumption by curtailing protein synthesis, gluconeogenesis, and lipogenesis (2, 3, 5, 30 –32).
The kinase therefore protects cells against death as a
consequence of energy depletion (3, 33, 34). Moreover,
AMPK inhibits cell proliferation (35). AMPK-sensitive ion
channels include the epithelial Na⫹ channel (ENaC; refs.
36 –39) and the delayed outwardly rectifying voltage gated
1
Abbreviations: ABR, auditory brainstem response; AMPK,
AMP-activated protein kinase; AT, acoustic trauma; BK, largeconductance voltage- and Ca2⫹-activated potassium; ENaC,
epithelial Na⫹ channel; IHC, inner hair cell; KCNQ1/
KCNE1, delayed outwardly rectifying voltage gated K⫹ channel; OHC, outer hair cell; P, postnatal day; PBS, phosphate
buffered saline; PFA, paraformaldehyde
0892-6638/12/0026-0001 © FASEB
These authors contributed equally to this work.
These authors contributed equally to this work.
3
Correspondence: Department of Physiology, University
of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.
E-mail: [email protected]
doi: 10.1096/fj.12-208132
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.
2
1
K⫹ channel (KCNQ1/KCNE1; ref. 40). The related saltinducible kinase decreases Na⫹/K⫹ ATPase activity (41).
AMPK is not only activated by energy depletion but,
in addition, by a decrease of oxygen (O2) levels (42),
exposure to nitric oxide (NO; ref. 43), and increasing
cytosolic Ca2⫹ activity (1). Thus, AMPK could participate in the regulation of Ca2⫹-sensitive ion channels.
The present study explored whether AMPK takes
part in the regulation of the large-conductance voltageand Ca2⫹-activated potassium (BK) channel or maxi-K⫹
channel. The BK channel is a heteromultimer composed of 4 ␣ and 4 ␤ subunits (44). The pore-forming
␣ subunit (KCNMA1), a member of the slo family of
potassium channels (45), had originally been identified
in Drosophila (46). The ␤1 subunit (KCNMB1) augments the voltage and calcium sensitivity of the ␣
subunit (47).
To determine the AMPK sensitivity of BK channels,
the K⫹ current was determined in Xenopus oocytes
expressing BK channels with or without additional
expression of wild-type AMPK (48), constitutively active
AMPK␥R70Q (49), and inactive AMPK␣K45R. Moreover,
the effect of AMPK coexpression on BK-channel protein abundance within the cell membrane was determined by confocal microscopy. The data indeed revealed a powerful up-regulation of the BK channel by
AMPK and AMPK␥R70Q, but not by AMPK␣K45R. To gain
insight into the in vivo relevance of AMPK-sensitive
BK-channel regulation, we studied the auditory system,
where BK channels are expressed among other cells in
inner and outer hair cells (IHCs and OHCs; ref. 50).
BK-channel-deficient mice show progressive hearing
loss (50, 51) and increased noise vulnerability (52). The
analysis of BK-channel abundance, hearing function,
and noise sensitivity of AMPK␣1-deficient and wild-type
mice (53) indeed revealed a profound role of AMPK in
the maintenance of BK-channel expression and susceptibility to auditory trauma.
MATERIALS AND METHODS
Voltage clamp in Xenopus oocytes
Xenopus oocytes were prepared as described previously (57, 58).
cRNA encoding the BK channel (20 ng) was injected with or
without 4.6 ng of cRNA encoding either AMPK␣1-HA ⫹ AMPK␤1Flag ⫹ AMPK␥1-HA (AMPKWT), or AMPK␣1-HA ⫹ AMPK␤1Flag ⫹ AMPK␥1R70Q-HA (AMPK␥R70Q) or AMPK␣1KDK45R-HA ⫹
AMPK␤1-Flag ⫹AMPK␥1-HA (AMPK␣K45R) on the day of preparation of the Xenopus oocytes. All experiments were performed at
room temperature 3 d after injection. In 2-electrode voltage-clamp
experiments, BK-channel currents were elicited every 20 s with 1-s
pulses from ⫺140 to ⫹190 mV applied from a holding potential
of ⫺60 mV. Pulses were applied in 20-mV increments. The data
were filtered at 1 kHz and recorded with a Digidata 1322A
A/D-D/A converter and Chart V.4.2 software for data acquisition
and analysis (Axon Instruments, Foster City, CA, USA; ref. 59). The
analysis of the data was performed with Clampfit 8 (Axon Instruments) software.
Immunocytochemistry of oocytes
Oocytes injected with the indicated cRNA were cryoprotected
in 30% sucrose and frozen in mounting medium. Cryosections with a thickness of 8 ␮m on coated slides were made and
stored at ⫺20°C. For immunostaining, sections were dehydrated at room temperature, fixed in 4% paraformaldehyde
for 15 min at room temperature, washed in TBS supplemented with 1% BSA and 0.5% Tween-20, and blocked for 1
h in 10% bovine serum in TBS (40). Sections were then
incubated with the primary rabbit anti-BK-channel antibody
(diluted 1:2000 in TBS supplemented with 1% BSA and 0.5%
Tween-20; ref. 51) in a moist chamber at 4°C overnight.
Subsequently, the sections were washed 5 times in TBS
supplemented with 1% BSA and 0.5% Tween-20. The binding
of the primary antibody was visualized with an anti-rabbit
FITC-conjugated antibody (diluted 1:1000 in TBS supplemented with 1% BSA and 0.5% Tween-20; Invitrogen,
Karlsruhe, Germany). The sections were incubated with the
secondary antibody for 1 h at room temperature and subsequently washed 5 times with TBS supplemented with 1% BSA
and 0.5% Tween-20. Stained oocyte sections were analyzed by
a fluorescence laser scanning microscope (LSM 510; Carl
Zeiss MicroImaging, Göttingen, Germany) with an A-Plan
⫻40/0.80 water-immersion objective. Brightness and contrast
settings were kept constant. The fluorescence images reflecting BK-channel membrane abundance were processed using
ZEN2009 software (Carl Zeiss MicroImaging).
Animals
Constructs
For generation of cRNA (54), constructs were used encoding
wild-type mouse BK channel (51), which was rendered Ca2⫹
insensitive by site-directed mutagenesis (BKM513I⫹⌬899 –903; ref.
55). The construct was kindly provided by J. Lingle (Washington
University School of Medicine, St. Louis, MO, USA). The
measurement of wild-type BK with the 2-electrode voltage clamp
requires increase in the intracellular Ca2⫹ level in oocytes, which
leads to likely side effects interfering with the measurement,
Thus, the Ca2⫹-insensitive mutant was used. Further constructs
used were wild-type AMPK (AMPK␣1-HA, AMPK␤1-Flag, and
AMPK␥1-HA; ref. 48), constitutively active AMPK␥1R70Q-HA
(49), and kinase-dead mutant AMPK␣1K45R-HA (39). The constructs were kindly provided by Scott Fraser (Burnet Research
Institute, Austin Health, Heidelberg, VIC, Australia) and Bruce
E. Kemp (St. Vincent’s Institute of Medical Research, University
of Melbourne, Fitzroy, VIC, Australia). All constructs were used
for the generation of cRNA as described previously (56).
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October 2012
Experiments were performed in adult AMPK␣1-deficient
(ampk⫺/⫺) and wild- type mice (ampk⫹/⫹). The age is indicated in the respective figure legend. The ampk⫺/⫺ mice have
been described previously (53). All animal experiments were
conducted according to the guidelines of the American
Physiological Society as well as the German law for the welfare
of animals and were approved by state authorities.
Hearing measurements and acoustic overexposure
As described previously (52, 60, 61), animals were anesthetized for determination of auditory brainstem responses
(ABRs) by intraperitoneal injection of 75 mg/kg body weight
ketamine hydrochloride (Ketavet 100; Pharmacia, Erlangen,
Germany), and 5 mg/kg body weight xylazine hydrochloride
(Rompun 290; Bayer, Leverkusen, Germany). For recording
of the ABR potentials, subdermal silver wire electrodes were
inserted at the vertex, ventrolateral to the measured ear
The FASEB Journal 䡠 www.fasebj.org
FÖLLER ET AL.
(active) and at the back of the mice (ground). Stimulus
generation and response recordings were accomplished using
a National Instruments Multi IO Card (NI PCI-6052; National
Instruments, Austin, TX, USA). Electrical signals were averaged over 64 –256 stimulus pairs (32–128 stimulus pairs in
case of f-ABR) after amplification (94 dB) and bandpass
filtering (0.2–5 kHz). The software averager included an
artifact rejection code (all waveforms with a peak voltage
exceeding a defined voltage were rejected) to eliminate the
ECG and muscle activity. Sound pressure was calibrated in situ
at all frequencies recorded prior to each measurement (microphone: Bruel & Kjaer 0.25-inch 4136; Nexus amplifier:
Bruel & Kjaer 2610; Bruel & Kjaer, Naerum, Denmark).
Recordings were performed in a sound-proof chamber (IAC,
Winchester, UK). The threshold was defined as the sound
pressure level where a stimulus-correlated response was
clearly identified in the recorded signal (52). Acoustic trauma
(AT) was induced by exposing anesthetized mice to free-field
band noise in a reverberating chamber (4 –16 kHz, 120 dB
SPL RMS for 1 h).
Cochelae tissue preparation
For RNA and protein isolation, cochleae were dissected,
immediately frozen in liquid nitrogen, and stored at ⫺80°C
before use (60). For immunohistochemistry, the cochleae
were fixed by immersion in 2% paraformaldehyde (PFA; all
chemicals from Sigma-Aldrich, Munich, Germany, unless
indicated otherwise) and 125 mM sucrose in 100 mM phosphate-buffered saline (PBS), pH 7.4, for 2 h. Cochleae were
decalcified after fixation for 15 min to 2 h in rapid bone
decalcifier (Eurobio; Fischer-Scientific, Nidderau, Germany).
After overnight incubation in 25% sucrose containing 1 mM
protease inhibitor in PBS (pH 7.4), cochleae were embedded
in optimal cutting temperature (O.C.T.) compound (Miles
Laboratories, Elkhart, IN, USA) and frozen at ⫺80°C. Tissues
were then cryosectioned at 10 ␮m thickness, mounted on
SuperFrost/plus microscope slides, dried for 1 h, and stored
at ⫺20°C before use. For whole-mount immunohistochemistry, cochleae were prepared and injected with 2% PFA for 15
min, followed by a dissection of the organ of Corti and cutting
it into 3 pieces: apical, medial and midbasal. Pieces were
mounted on coverslips with Cell Tak (Becton Dickinson,
Bedford, MA, USA). After fixation, tissue was treated as
described for cochlear cryosections.
Single hair cell isolation and cDNA synthesis
Apical and medial half-turns of the organ of Corti of 25-d-old
[postnatal day 25 (P25)] wild-type mice were dissected and
fixed on a coverslip. IHCs and OHCs were separately harvested with micropipettes (⬃30 IHCs and 80 OHCs; refs. 62,
63) under fast flow of a physiological solution with partial
replacement of Cl⫺ by gluconate, which better preserved the
hair cells (120 mM Na-gluconate, 35 mM NaCl, 4.8 mM KCl,
1.3 mM CaCl2, 0.9 mM MgCl2, 0.7 mM NaH2PO4, 10 mM
HEPES, and 5.6 mM glucose; pH 7.35; 320 mosmol/kg). Cells
were immediately frozen in liquid nitrogen, and cDNAsynthesis was performed with Superscript III (Invitrogen)
following the manufacturer’s manual.
RT-PCR
For RT-PCR analysis, mRNA from 42-d-old (P42) mouse
cochleae was extracted by means of Dynabeads (Dynal, Oslo,
Norway) following the manufacturer’s instructions. After reverse transcription using iScript reverse transcriptase (BioRad, Munich, Germany), oligo-dT, and random primers, an
AMPK-SENSITIVE BK-CHANNELS
AMPK␣1 fragment of wild-type mice was amplified by RT-PCR
using TaqDNA polymerase (Qiagen, Hilden, Germany) with
the oligonucleotides 5=-TGAGAACGTCCTGCTTGATG-3=
and 5=-CCGAGTTAAATGGTGGTCGT-3= (annealing temperature 55°C; 35 cycles for cochlea, 50 cycles for hair cells). For
the cochlea and OHCs, a nested PCR was performed with the
oligonucleotides 5=-GCTGTGGCTCACCCAATTAT-3= and 5=TCCTCCGAACACTCGAACTT-3= (annealing temperature
55°C; 35 cycles). After nested PCR, a 397-bp fragment was
amplified. The resulting PCR products were analyzed on
agarose gels stained by ethidium bromide. All PCR experiments were done at least in triplicate. The PCR product
obtained from cochlea cDNA was sequenced by GATC (Konstanz, Germany).
Immunohistochemistry of cochleae
For immunohistochemistry, cochlear sections were defrosted
and permeabilized with 0.5% Triton X-100 for 10 min at
room temperature, preblocked with 4% normal goat serum
in PBS, and incubated overnight at 4°C with the primary
antibody. As primary antibody, mouse (for cryosections) and
rabbit (for whole mount) monoclonal anti-BK␣ (mouse: 1:50,
75-022, NeuroMab, University of California/U.S. National
Institutes of Health, Davis, CA, USA; and rabbit: 1:50, APC021, Alomone Labs, Jerusalem, Israel) and mouse monoclonal anti-neurofilament (NF200, 1:8000; N5139; Sigma-Aldrich) were used. The primary antibodies were detected with
fluorescence-labeled secondary IgG antibodies (Alexa Fluor
488-conjugated antibody, Molecular Probes, Eugene, OR,
USA; or Cy3-conjugated antibody, Jackson ImmunoResearch
Laboratories, West Grove, PA, USA). Sections were embedded with Vectashield mounting medium, DAPI stained for
cell nuclei (Vector Laboratories, Burlingame, CA, USA) and
photographed using an Olympus AX70 microscope equipped
with epifluorescence illumination and a motorized z axis
(Olympus, Tokyo, Japan). Images were acquired using a CCD
camera and the imaging software CellF (OSIS GmbH, Münster, Germany). Figure 6E, H shows composite images that
represent the maximum intensity projection over all layers of
the z stack.
Western blot
For Western blot analysis, cochlear tissue from AMPK␣1deficient (ampk⫺/⫺) mice and age-matched control mice
(ampk⫹/⫹) was homogenized, and Western blot analysis was
performed as described previously (64). Blotted proteins
were incubated with rabbit polyclonal AMPK␣1 antibody
(1:1000; NB-100-239; Novus Biologicals, Cambridge, UK),
which detects a band of approximately 64 kDa. Furthermore,
a mouse monoclonal BK-channel antibody (1:500; 75-022;
NeuroMab) that recognizes a band of ⬃112 kDa and, for
semiquantitative detection, a mouse monoclonal Ezrin antibody (1:400, DLN-10378; Dianova GmbH, Hamburg, Germany), which detects a band of 81 kDa, were used, followed
by incubation with an ECL peroxidase-labeled anti-mouse
(1:2000; NA931; GE Healthcare, Little Chalfont, UK) or
anti-rabbit (1:2000; NA934; GE Healthcare) antibody. Labeled proteins were detected by chemiluminescence using
the ECL Plus Western blotting detection reagents from
Amersham Biosciences (Freiburg, Germany). For densitometric analysis, ImageJ 1.44 (U.S. National Institutes of Health,
Bethesda, MD, USA) was used.
Statistical analysis
Data are provided as means ⫾ se, n represents the number of
experiments, and, in case of the ABR measurements, n
3
represents the number of animals. All oocyte experiments
were repeated with ⱖ3 batches of oocytes. In all repetitions,
qualitatively similar data were obtained. The magnitude of
the current and the effect of AMPK could vary between
different batches of oocytes. Therefore, the currents were
normalized. Data were tested for significance using ANOVA.
For statistical analysis of click-ABR data, Student’s t test was
used in case of genotype comparisons; for comparisons
involving repeated measurements, 1-way ANOVA adapted for
repeated measurements with Tukey’s multicomparison test as
posttest was used. For frequency-specific ABR data comparisons, 2-way ANOVA adapted for repeated measurements with
Tukey’s multicomparison test as posttest (Prism 2.01; GraphPad, San Diego, CA, USA) was used to control for an inflating
␣ error. For all experiments, values of P ⬍ 0.05 were
considered statistically significant.
RESULTS
AMPK sensitivity of BK currents
In a first step, calcium-insensitive BK channels were
heterologously expressed in Xenopus oocytes with and
without coexpression of wild type AMP-activated protein kinase (AMPK␣1-HA ⫹ AMPK␤1-Flag ⫹ AMPK␥1HA), and K⫹ currents (IBK) were quantified using the
dual-electrode voltage clamp. As shown in Fig. 1A,
depolarization to ⫹190 mV triggered an outward current in oocytes expressing BK channels and, to a lesser
extent, in water-injected oocytes. More important, the
depolarization-induced current IBK was significantly
enhanced by coexpression of wild-type AMPK (Fig. 1A,
B). The current-voltage dependence is displayed in Fig.
1C. Figure 1D shows the current-voltage relationship
after substracting the current determined in waterinjected oocytes.
In a second step, experiments were performed to
determine whether the effect of AMPK was mimicked
by constitutively active AMPK␥R70Q (AMPK␣1-HA ⫹
AMPK␤1-Flag ⫹ AMPK␥1R70Q-HA) and/or the kinasedead mutant AMPK␣K45R (AMPK␣1K45R ⫹ AMPK␤1 ⫹
AMPK␥1). As shown in Fig. 2, the coexpression of
AMPK␥R70Q in BK-expressing oocytes similarly increased IBK. In contrast, AMPK␣K45R did not significantly modify IBK in BK-channel-expressing oocytes.
Thus, kinase activity was required for the stimulatory
effect of AMPK on IBK. The current tended to be higher
in oocytes expressing BK together with wild-type AMPK
than in oocytes expressing constitutively active AMPK; a
difference, however, not reaching statistical significance.
An increase in IBK could have resulted from an
increase in BK-channel protein abundance in the cell
membrane. To test this possibility, BK-channel protein
abundance was determined by immunocytochemistry
in Xenopus oocytes injected with water, in oocytes
expressing BK channel alone, and in oocytes expressing
BK channel together with either wild-type AMPK, constitutively active AMPK, or the inactive mutant of
AMPK. As apparent from confocal microscopy (Fig. 3),
Figure 1. Coexpression of AMPK increased the K⫹
current in BK-expressing Xenopus oocytes. A) Original
tracings of the current from ⫺150 to ⫹190 mV in
Xenopus oocytes injected with water (a), expressing BK
channels alone (b), or expressing BK channels with
additional coexpression of wild-type AMPK (c). B) Arithmetic means ⫾ se (n⫽37– 67) of the normalized K⫹
current at ⫹190 mV in Xenopus oocytes injected with
water (open bar), expressing BK channels alone
(shaded bar), or expressing BK channels with wild-type
AMPK (solid bar). Average current of oocytes expressing BK channels alone at ⫹190 mV was 1292 ⫾ 90 nA
(n⫽67). ***P ⬍ 0.001 vs. BK alone; ANOVA. C) Current
(I)–voltage (V) curves of the data as in B. D) I-V curves
of the data as in B after substracting the current
determined in Xenopus oocytes injected with water.
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FÖLLER ET AL.
3
norm. current [arb. Units]
2.5
2
***
+
**
BK
1.5
1
0.5
0
AMPK
AMPKK45R
AMPKR70Q
Figure 2. Constitutively active AMPK␥R70Q but not inactive
AMPK␣K45R increased the current in BK-channel-expressing
Xenopus oocytes. Arithmetic means ⫾ se (n⫽10 – 43) of the
normalized K⫹ current at ⫹100 mV in Xenopus oocytes
injected with water (1st bar), expressing BK channels alone
(2nd bar) or expressing BK channels with additional coexpression of wild-type AMPK (3rd bar), of kinase-dead mutant
AMPK␣K45R (4th bar) or of constitutively active AMPK␥R70Q
(5th bar). Difference between wild-type AMPK and constitutively active AMPK␥R70Q is not statistically significant
(P⬎0.05). **P ⬍ 0.01, ***P ⬍ 0.001 vs. BK alone.
the cell surface expression of the BK-channel protein in
Xenopus oocytes injected with cRNA encoding BK channel
was indeed increased by constitutively active AMPK and,
to a lesser extent, by wild type AMPK but not by
AMPKK45R. Quantification of the confocal images resulted in a relative density of BK in the oocyte membrane
of 32 ⫾ 2 arbitrary units (AU; n⫽12) following BK
expression alone, of 44 ⫾ 3 AU (n⫽12) following coexpression of AMPK, of 65 ⫾ 3 AU (n⫽12) following
coexpression of AMPK␥R70Q and of 31 ⫾ 1 AU (n⫽12)
following coexpression of AMPK␣K45R. Thus, AMPK
(P⬍0.05) and AMPK␥R70Q (P⬍0.001), but not
AMPK␣K45R, significantly increased protein abundance in
Xenopus oocytes. In addition, the effect of AMPK␥R70Q on
BK surface expression was significantly more pronounced
(P⬍0.001) than that of AMPK.
To test whether AMPK further modifies BK kinetics,
the macroscopic BK-channel kinetics were determined.
The activation can be fitted to 2 and the deactivation to
1 exponential. As a result, coexpression of AMPK,
AMPK␥R70Q, or AMPKK45R did not significantly modify
the time constants for the fast activation, the slow
activation, and the deactivation (Supplemental Fig. S1).
AMPK␣1 expression in the cochlea
To explore the in vivo significance of AMPK in the
regulation of BK expression, we chose the auditory
system, where BK is important for noise protection in
OHCs (50, 52). To analyze the expression of AMPK␣1
in the murine cochlea, we performed RT-PCR and
nested RT-PCR from cochlear mRNA (P42) and mRNA
from isolated IHCs and OHCs (OHCs, P21; IHCs, P35)
of wild-type mice. The expected PCR product with a
size of 397 bp for AMPK␣1 was amplified in cochlear
AMPK-SENSITIVE BK-CHANNELS
tissue (Fig. 4A, Co) and isolated OHCs (Fig. 4A) but not
in isolated IHCs. Otoferlin, a protein that is selectively
expressed in IHCs of the cochlea (64), was used as
positive control. To specify the AMPK␣1 protein expression in cochlear tissue of adult ampk⫹/⫹ mice,
Western blot analysis (Fig. 4B) was performed using a
rabbit polyclonal AMPK␣1-specific antibody. A polypeptide band of the appropriate expected size of 63
kDa could be detected in tissue of ampk⫹/⫹ mice,
whereas no signal in tissue of ampk⫺/⫺ mice was found
(Fig. 4B).
Noise vulnerability of AMPK␣1-deficient mice
To determine whether AMPK is of functional importance for maintenance of hearing, adult ampk⫹/⫹ and
ampk⫺/⫺ mice were investigated on analysis of ABR
thresholds (Fig. 5A, B). The hearing thresholds to
click-specific (Fig. 5A) and frequency-specific auditory
stimuli (Fig. 5B) were not significantly different between ampk⫹/⫹ and ampk⫺/⫺ mice (Fig. 5A, B, pretest).
Acoustic overstimulation (stimulation with a noise of
120 dB SPL, 4-16 kHz bandwidth for 1 h) resulted in a
significant and permanent increase in the hearing
thresholds of both genotypes. Immediately after the
exposure, the hearing threshold was similar in ampk⫹/⫹
and ampk⫺/⫺ mice (Fig. 5A, after AT). However, recovery from AT was impaired in ampk⫺/⫺ mice; i.e., 7 d
after exposure, the hearing loss was significantly higher
in ampk⫺/⫺ mice than in ampk⫹/⫹ mice for both clickand frequency-specific ABR (Fig. 5A, B, 7 d after AT).
This indicates that AMPK has a profound effect on the
noise vulnerability of mice.
BK-channel expression in ampkⴚ/ⴚ mice and
ampkⴙ/ⴙ mice
To explore whether the increased noise vulnerability of
ampk⫺/⫺ mice is indeed paralleled by altered AMPKdependent regulation of BK, Western blotting and immunohistochemistry were performed. For Western blot analysis (Fig. 6A, B), a mouse monoclonal antibody directed to
the amino acid sequence 690 –1196 of the slo1 subunit of
BK protein (51) was found to crossreact with a polypeptide of the expected size of ⬃112 kDa in the cochlea of
adult ampk⫹/⫹ mice (Fig. 6A). This expression was drastically lower (6.2⫾0.7% of wild-type expression, tissue from
n⫽5 animals/genotype) in ampk⫺/⫺ mice (Fig. 6A). The
reduction was semiquantified on parallel detection of
Ezrin, using a mouse monoclonal Ezrin antibody, which
crossreacts with a polypeptide of the expected size of 81
kDa. When the BK protein was stained in OHCs of the
ampk⫺/⫺ mouse cochlea (Fig. 6F, G), the expression
typical of the basal pole of OHCs in ampk⫹/⫹ mice (Fig.
6C, D) was reduced, in particular in the high-frequency
regions of the cochlea (Fig. 6C, F, shown for the midbasal
cochlea turn). In IHCs, BK was found to be expressed at
the supranuclear level of IHC (Fig. 6B, D, E, G) in both,
mutant and wild-type mice. Apical OHCs of ampk⫺/⫺ mice
were not affected (data not shown). The expression of BK
5
Figure 3. Coexpression of AMPK increased the BK-channel protein abundance within the plasma membrane of oocytes.
Confocal images of BK-channel protein abundance in the plasma membrane of Xenopus oocytes injected with water (top left
panel), injected with cRNA encoding constitutively active AMPK␥R70Q (top middle panel), injected with cRNA encoding BK
channels without (top right panel) or with additional coexpression of wild-type AMPK (bottom left panel), of kinase-dead
mutant AMPK␣K45R (bottom middle panel) or of constitutively-active AMPK␥R70Q (bottom right panel). Cells were subjected to
immunofluorescence staining using a FITC-conjugated antibody (green). Scale bars ⫽ 20 ␮m.
in OHCs following auditory trauma was furthermore
reduced and correlated with a more pronounced loss of
OHCs in the respective regions (data not shown).
DISCUSSION
The present study reveals a novel function of the AMPactivated kinase AMPK. The kinase up-regulates the Ca2⫹sensitive large-conductance BK channel. As AMPK is
activated by an increase in cytosolic Ca2⫹ activity (1),
AMPK is expected to contribute to the up-regulation of
BK channels by increased cytosolic Ca2⫹ levels.
As illustrated in Fig. 1, BK-channel activity is highly
sensitive to voltage. The current is small at a cell
membrane potential below ⫺90 mV but increases
steeply at positive voltages. Thus, the channel accomplishes K⫹ exit primarily in the depolarized state. On
coexpression of AMPK, less depolarization is required
for the accomplishment of any given K⫹ exit.
The study did not attempt to elucidate the mechanisms involved in AMPK-dependent regulation of BK
channels. AMPK has most recently been shown to
6
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October 2012
up-regulate Kv2.1 by direct phosphorylation of the
channel protein (65) AMPK has further been shown to
up-regulate KATP channels (66) and TRPC3 channels
(67) by direct interaction with the channel protein
fostering channel insertion into the cell membrane
(67). The present study reveals that AMPK regulates BK
channels at least in part by modifying the protein
abundance within the cell membrane. In theory, AMPK
may be effective by stimulating channel expression, by
facilitating channel insertion into the membrane, or by
decreasing channel retrieval from the cell membrane.
However, AMPK may down-regulate ion channels (40,
68 –72) by stimulation of the ubiquitin ligase Nedd4-2,
which ubiquitinates the channels, thus preparing them
for proteasomal degradation (36 –38). AMPK may further down-regulate ion channels by fostering the formation of PIP2 (69). The effects of AMPK and
AMPK␥R70Q were not identical to the effects on current.
However, as the currents were determined in batches of
oocytes other than those used for protein abundance,
no safe comparisons can be made between the effect of
AMPK on current and protein abundance. It is, however, safe to conclude that AMPK is at least partially
The FASEB Journal 䡠 www.fasebj.org
FÖLLER ET AL.
Figure 4. AMPK␣1 expression in the cochlea. A) Nested PCR
detecting AMPK␣1 mRNA in wild-type mouse cochleae (Co;
P19, 397 bp) and in isolated OHCs (P21, 397 bp) but not in
isolated IHCs (P35). Otoferlin (207 bp) was used as positive
control. B) Western blot analysis showing AMPK␣1 expression in 3-mo-old cochleae tissue. A specific band at the
expected 63 kDa could be detected in ampk⫹/⫹ tissue,
whereas no band was seen in ampk⫺/⫺ tissue.
effective through up-regulation of BK-channel protein
abundance in the cell membrane.
Stimulation of BK channels by AMPK is presumably a
double-edged sword. On the one hand, activation of K⫹
channels enhances the driving force for Na⫹-coupled
transport of glucose and other substrates and at the
same time drives electrogenic HCO3⫺ exit fostering
cytosolic acidification, which, in turn, would stimulate
the Na⫹/H⫹ exchanger (73). Thus, hyperpolarization
augments Na⫹ entry and thus increases the requirement for energy-consuming Na⫹ extrusion by the
Na⫹/K⫹ ATPase (73). Stimulation of K⫹ channels may
further foster cellular K⫹ loss during impaired function
of Na⫹/K⫹ ATPase in energy-depleted cells. Cellular
K⫹ loss may, in turn, trigger suicidal cell death (74 –78).
The increased HCO3⫺ exit with subsequent cytosolic
acidification in hyperpolarized cells could accelerate
the death of apoptotic cells (79) and compromise
glycolysis (80). Possibly to counteract hyperpolarization
and cellular K⫹ loss, AMPK inhibits the activity of
KCNQ1KCN/E1 (40) and of Kir2.1 (72). However,
stimulation of K⫹ channels results in hyperpolarization
of the cell membrane, fostering Cl⫺ exit and thus
counteracting potentially harmful cell swelling (81, 82).
In excitable cells, stimulation of K⫹ channels decreases
activation of voltage-gated Ca2⫹ channels and thus
excitability, thus being cell protective.
In the inner ear, the Na⫹/K⫹ ATPase (83– 85) may
similarly be compromised by energy depletion in noise
damage. However, BK channels protect against noise
damage. BK channels are indeed expressed in IHCs
(86 –95, 95, 96), OHCs (50, 52, 96 –98) and efferent
fibers to OHCs (99). BK channels in IHCs have been
postulated to play a role for graded receptor potentials
and phase locking (86, 89, 100), with an expected
profound effect on hearing. However, normal hearing
thresholds were detected in young mutants with deleted BK channels, and the predicted function of BK
channels in IHCs needed to be corrected. The role of
BK channels in IHCs is still not well understood. BK
channels in IHCs obviously play an essential role for the
precise timing of high-frequency cochlear signaling in
IHCs, as well as in the primary afferent neurons, rather
than for basic functions on IHCs (101). The same holds
true for BK-channel expression in OHCs, at least at
Figure 5. AMPK deficiency increased noise vulnerability after acoustic overstimulation. A) Means ⫾ se (n⫽6) of the ABR
thresholds for a click stimulus in 2- to 4-mo-old (average 92 d) ampk⫺/⫺ mice and ampk⫹/⫹ mice prior to (left bars), immediately
after (middle bars) and 7 d after (right bars) an acoustic overstimulation (AT, exposure to 4 –16 kHz, 120 dB SPL RMS for 1
h). Both genotypes exhibit a statistically significant hearing loss (P⬍0.001, after AT vs. pretest), and a statistically significant
recovery (P⬍0.01 for ampk⫺/⫺ and P⬍0.001 for ampk⫹/⫹, 7 d after AT vs. after AT) using 1-way ANOVA adapted for repeated
measurements. **P ⬍ 0.01. B) Means ⫾ se (n⫽6) of the frequency-specific hearing thresholds (2– 45.25 kHz) in 2- to 4-mo-old
(average 92 d) untreated ampk⫹/⫹ mice (solid gray line) and ampk⫺/⫺ mice (dashed gray line) (not significantly different,
P⬎0.05; ANOVA), and 7 d after an acoustic overstimulation of ampk⫹/⫹ mice (solid black line) and ampk⫺/⫺ mice (dashed black
line). Hearing loss in both genotypes was significantly different (P⬍0.001; ANOVA).
AMPK-SENSITIVE BK-CHANNELS
7
Figure 6. BK expression in the
cochlea of AMPK␣1 wild-type
and knockout mice. A) Western
blot analysis displaying the differences in BK expression between
AMPK␣1 wild-type (ampk⫹/⫹)
and AMPK␣1-deficient mice
(ampk⫺/⫺). In ampk⫹/⫹ cochleae,
a BK-specific band at the expected 112 kDa could be detected, whereas almost no signal
(6.2⫾0.7% of wild-type expression; tissue from n⫽5 animals/
genotype) was found in ampk⫺/⫺
tissue. An anti-Ezrin antibody was
used as loading control (81 kDa). B–G) Immunohistochemistry on mature cochlea cryosections (B, C, E, F) and whole-mount (D, G)
and with a BK-specific antibody (green in cryosections, red in whole mount). BK was expressed in midbasal IHCs (B, E) and OHCs
(C, F) of AMPK␣1 wild-type mice (B–D, ampk⫹/⫹). AMPK␣1-deficient mice (E–G, ampk⫺/⫺) exhibited less BK expression in OHCs (F,
G). However, the IHCs (E, G) showed a normal BK expression pattern. NF200 (green) was used to stain nerve fibers for better
orientation. Slices were counterstained with DAPI (blue) to highlight nuclei. Scale bars ⫽ 10 ␮m (B, C, E, F); 25 ␮m (D, G).
younger age. The analysis of mice lacking functional
BK␣ (KCNMA1) channels yielded normal tuning
curves until ⬃4 wk of age, followed by a slowly progressive hearing loss mostly affecting high frequencies. The
progressive hearing loss goes hand in hand with a loss
of surface expression of KCNQ4 (50), due to lack of
BK-channel-dependent counteraction against noise-induced Ca2⫹ overload of the cells (52).
A hearing loss 7 d following acoustic overexposure
similar to that in ampk⫺/⫺ mice has been observed in
mice lacking cGMP-dependent protein kinase type I
(cGKI; ref. 102). Similar to AMPK, cGKI directly influences BK channels (103, 104) presumably preventing
noise-induced Ca2⫹ overload by targeting BK channels
(102). The decreased BK-channel protein abundance
in the cochlea in the absence of AMPK and its partial
reduction in OHCs surface suggests that AMPK protects
OHCs from noise damage at least partially by stimulation of K⫹ channels with subsequent decrease of OHC
excitability. Lack of AMPK may result in decreased
BK-channel surface expression followed by noise-induced Ca2⫹ overload. The protective effect of AMPK
may involve further mechanisms, such as up-regulation
of facilitative glucose carriers with subsequently increased cellular glucose uptake (6, 12, 13, 15–18, 21,
24 –26, 105). Thus, regulation of BK channels contributes to, but does not necessarily fully account for, the
protective role of AMPK against noise-induced hearing
loss.
In theory, AMPK-dependent regulation of BK channels may similarly influence cell survival and/or function in further organs, such as the retina (106), brain
(107–110), heart (111), endothelial cells (112), vascular smooth muscle (113, 114), airway smooth muscle
(115, 116), kidney (117), and pancreatic ␤ cells (118).
AMPK-dependent regulation of BK channels may further promote survival of tumor cells (119).
In summary, energy-sensing AMPK is a powerful
stimulator of BK channels. At least partially due to
up-regulation of BK channels, AMPK decreases the
vulnerability of OHCs to noise.
8
Vol. 26
October 2012
The authors gratefully acknowledge the generosity of
Benoit Viollet (Institut Cochin, Université Paris Descartes,
Centre National de la Recherche Scientifique Unité Mixte de
Recherche 8104, Paris, France), who provided the AMPK␣1deficient mice. The authors acknowledge the experimental
support of Ioana Alesutan and Wibke Singer and the technical assistance of E. Faber. The manuscript was meticulously
prepared by L. Subasic. Isolated hair cells were kindly provided by J. Engel (University of Tübingen; current address:
Saarland University, Homburg/Saar, Germany). This study
was supported by the Deutsche Forschungsgemeinschaft and
the Interdisziplinäres Zentrum für Klinische Forschung of the
University of Tübingen (Nachwuchsgruppe to M.F.).
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Received for publication March 19, 2012.
Accepted for publication June 20, 2012.
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