From the Membrane Biology Group, Department of Physiology, Medical
School, Teviot Place, University of Edinburgh, Edinburgh, Scotland, EH8
9AG, United Kingdom and the Institut Fuer Biochemische
Pharmakologie, Peter Mayr-Strasse 1, A-6020 Innsbruck, Austria
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ABSTRACT |
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Adrenal glucocorticoids exert powerful effects on
cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact
mouse anterior pituitary corticotrope (AtT20) cells
glucocorticoid-induced proteins render large conductance
calcium-activated potassium (BK) channels insensitive to inhibition by
protein kinase A (PKA). In this study we have addressed whether this
action of glucocorticoids is mediated via protein phosphatase activity
at the level of single BK channels. In isolated inside-out patches from
control AtT20 cells BK channels (125 pS) were inhibited by activation
of closely associated PKA. Pretreatment (2 h) of cells with 1 µM dexamethasone before patch excision did not
modify the intrinsic properties or expression levels of BK channel
-subunits in AtT20 cells. However, PKA-mediated inhibition of BK
channel activity in isolated patches from steroid-treated cells was
severely blunted. This effect of steroid was not observed using
adenosine 5'-O-(3-thiotriphosphate) as phosphate donor or
on exposure of the intracellular face of the patch with 10 nM of the protein phosphatase inhibitors okadaic acid or
calyculin A but was mimicked by application of protein phosphatase 2A
(PP2A) to the intracellular face of patches from control cells.
Glucocorticoids did not modify total PP2A activity in AtT20 cells,
suggesting that modified PP2A-like phosphatase activity closely
associated with BK channels is required for glucocorticoid action.
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INTRODUCTION |
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Glucocorticoid hormones exert profound effects on cellular excitability in endocrine and nerve cells through regulation of ion channel activity that requires the rapid induction of new proteins (1-3). Increasing evidence suggests that potassium channels are major targets for glucocorticoid action. Although glucocorticoids rapidly induce potassium channel subunits in some systems (4, 5), the mechanisms of channel regulation by glucocorticoids in endocrine cells and neurons are largely not understood (1-3).
Anterior pituitary corticotrope cells have been widely used as a physiologically relevant model system to explore the mechanisms of early glucocorticoid action (6, 7). In the mouse corticotrope cell line, AtT20 D16:16, the cAMP-mobilizing neuropeptide, corticotrophin-releasing factor, stimulates adrenocorticotropin secretion through the concerted action of protein kinase A (PKA)1 to activate L-type calcium channels and inhibit BK channels and subsequent enhancement of calcium influx through L-type calcium channels (3, 8). In turn, glucocorticoids rapidly (within 2 h) inhibit corticotropin-releasing factor-stimulated secretion through the induction of new proteins (9, 10). We have previously demonstrated in metabolically intact AtT20 D16:16 corticotropes that glucocorticoid-induced proteins render BK channels insensitive to inhibition by protein kinase A and that the action of the steroid is central for the early inhibition of adrenocorticotropin hormone secretion in this system (3). Intriguingly glucocorticoids also block protein kinase A-mediated inhibition of calcium-activated potassium channels underlying the slow after-hyperpolarization in hippocampal neurons (1, 11), suggesting that calcium-activated potassium channels are common targets for reciprocal regulation of cellular excitability by glucocorticoid-induced proteins and cAMP-dependent phosphorylation.
In corticotropes, glucocorticoid-induced proteins may modulate other signaling pathways to regulate BK channel activity or may directly modulate BK channel function themselves. Increasing evidence suggests that the activity of BK channels are dynamically regulated by the interaction of protein kinases and phosphatases intimately associated with the channel complex (12-15). Because glucocorticoids specifically antagonize PKA-mediated regulation of BK channels but not L-type calcium channels in AtT20 D16:16 cells (3), we hypothesized that glucocorticoids may exert their effects through protein phosphatase activity at the level of the BK channel complex itself. Indeed increasing evidence from other systems suggests that glucocorticoids mediate some of their effects through regulation of serine/threonine as well as tyrosine-protein phosphatase activity (16-19).
Inhibition of protein phosphatases modulates cAMP accumulation and metabolism in intact AtT20 D16:16 cells (20) and glucocorticoid receptor function in many cell types (21) thus precluding definitive analysis of the mechanism of glucocorticoid action at the level of BK channels themselves in intact cells. Thus to directly address whether protein phosphatases are involved in the ability of glucocorticoids to block PKA-mediated inhibition of BK channel activity we have examined the regulation of BK channel activity in excised inside-out patches from control and glucocorticoid-pretreated AtT20 D16:16 corticotropes. The data in this report demonstrate that glucocorticoid regulation of BK channel activity requires protein phosphatase 2A activity closely associated with the BK channel complex.
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EXPERIMENTAL PROCEDURES |
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AtT20 D16:16 Cell Culture-- Clonal mouse anterior pituitary (AtT20 D16:16, passage 18-32) cells were maintained as described previously (3) and used 3-7 days post-plating on glass coverslips. Cells were treated with 1 µM of the synthetic glucocorticoid dexamethasone or vehicle (<0.01% Me2SO) for 2 h at 37 °C in serum-free Dulbecco's modified Eagle's medium, pH 7.4, buffered with 25 mM HEPES and containing 0.25% bovine serum albumin. Cells were then transferred to the bath solution (dexamethasone-free) outlined below for electrophysiological recording. Regulation of single channel events in isolated inside-out patches from control or dexamethasone-treated cells was performed in parallel on the same passage of cells to avoid potential intra-passage variations in responsiveness.
Electrophysiology--
Single BK channel events were recorded in
the inside-out patch configuration of the patch clamp technique using
physiological potassium gradients. The bath (intracellular face of
patch) solution contained in (mM): 140 KCl, 1 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetracetic acid, 10 HEPES, 30 glucose, 1 ATP, pH 7.35, and the respective concentrations of Mg2+ and Ca2+ as indicated in
the figure legends. The patch pipette (extracellular face of patch)
contained in (mM): 140 NaCl, 5 KCl, 5 MgCl2,
0.1 CaCl2, 10 HEPES, 20 glucose, pH 7.4, containing 0.002 tetrodotoxin. Single channel events were recorded for 20-30 s every
2-5 min at the voltages indicated in the figure legends. Preliminary
stability plot experiments demonstrated that BK channel activity was
stable for >1 h under the recording conditions used (data not shown). Data acquisition and voltage protocols were controlled by an Axopatch 200B amplifier and pCLAMP 6 software (Axon Instruments Inc., Foster City, CA). Pipettes were manufactured from Garner 7052 glass, sylgarded, with resistances of 1-3 M in physiological saline after
fire polishing.
Western Blotting--
Crude membrane homogenates from AtT20
D16:16 cells were prepared by homogenizing ~107 cells on
ice in homogenization buffer (in mM): 50 Tris-HCl, pH 7.4, 140 KCl, 1 EGTA, 1 MgCl2 containing 12 units/ml aprotinin, 5 µg/ml leupeptin, 6 mM
4-(2-aminoethyl)benzenesulfonylfluoride, and 4 mM pepstatin
A followed by two freeze thaw cycles. After centrifugation for 5 min
1000 × g at 4 °C the resultant supernatant was
pelleted at 20,000 × g to give the crude membrane
fraction. Protein samples (15 µg) were separated on a 10% SDS gel
and electroblotted to Immobilon polyvinylidene difluoride membranes.
Membranes were blocked for 2 h at room temperature with PBS
containing 0.1 mM EDTA, 0.1% Triton X-100, pH 7.4, (PBS-TE) and 5% (w/v) low fat milk (Marvel). Blots were incubated
overnight at 4 °C with a 1:2000 dilution of the affinity purified
antibody slo(913-926) (directed toward residues
913-926 of the pore-forming
-subunit of mouse brain BK channels;
Ref. 22) in PBS-TE containing 1% (w/v) Marvel. Blots were washed five
times with PBS-TE and incubated for 45 min at room temperature with
horseradish peroxidase-labeled anti-rabbit IgG (Amersham Pharmacia
Biotech, 1:5000 final dilution) in PBS-TE containing 5% (w/v) Marvel.
After five washes in PBS-TE, blots were incubated with Amersham
Pharmacia Biotech ECL reagents according to the manufacturer's
protocol, and blots were exposed to ECL film in the linear response
range (Amersham Pharmacia Biotech).
Protein Phosphatase Assays--
Protein phosphatase activity of
crude cytosolic and membrane fractions were determined by using the
molybdate:malachite green:phosphate complex assay using the synthetic
phosphopeptide RRA(pT)VA as substrate essentially as described by the
manufacturer (Promega Corporation, Madison, WI). Cytosolic and crude
membrane fractions were prepared from control and dexamethasone-treated
AtT20 D16:16 cells as for Western blotting in homogenization buffer (in
mM): 50 Tris-HCl, pH 7.4, 140 KCl, 1 EGTA, 1 MgCl2 containing 12 units/ml aprotinin, 5 µg/ml
leupeptin, 6 mM 4-(2-aminoethyl)benzensulfonylfluoride, and 4 mM pepstatin A. To remove endogenous phosphate,
cytosolic fractions were passed twice through a 10-ml bed volume of
Sephadex G-25, resuspended membrane fractions were incubated for 20 min at 4 °C with 10 volumes of Sephadex G-25, and the 200 × g supernatant was washed and pelleted twice at 20,000 × g in Tris-HCl as above. Phosphatase assays were performed
in a volume of 50 µl in imidazole buffer (in mM: 50 imidazole, pH 7.2, containing 0.2 EGTA, 0.02% (v/v)
-mercaptoethanol, and 0.1 mg/ml bovine serum albumin) for 30 min at
30 °C using 100 µM of RRA(pT)VA as substrate. PP2A
activity was determined as the difference in total phosphatase activity and phosphatase activity in the presence of 10 nM okadaic
acid. Under the conditions used >80% of phosphatase activity was
sensitive to 10 nM okadaic acid. Reaction was terminated by
addition of the molybdate dye buffer and incubated for 30 min at room
temperature, and absorbance was determined at 600 nm.
Reagents--
Purified protein phosphatase 2A catalytic subunit
and reagents for PP2A activity assay were from Promega Corporation
(Southampton, UK). Calyculin A, okadaic acid, and norokadone were from
LC Laboratories (Alexis Corporation Ltd., Nottingham, UK). The specific
protein kinase A inhibitor peptide (PKI(5-24)) was from
Sigma or Calbiochem-Novabiochem Ltd. (Nottingham, UK). Tetrodotoxin was from Calbiochem-Novabiochem Ltd. (Nottingham, UK). Polyvinylidene difluoride membranes and reagents for SDS-polyacrylamide gel
electrophoresis and Western blotting were from Bio-Rad Laboratories,
Ltd. (Hertfordshire, UK). All other reagents were from Sigma or
BDH-Merck (Poole, Dorset, UK). Dexamethasone was stored at 20 °C
at 10 mM in Me2S0.
Statistics-- Data are expressed as the means ± S.E. Statistical significance was determined by Student's t test for paired and unpaired data as appropriate. A p value of less than 0.05 was considered to be significant.
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RESULTS |
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In inside-out patches from control AtT20 D16:16
corticotropes single BK channel events were characterized by their
slope conductance (125 ± 3 pS in physiological potassium
gradients and 2 mM intracellular magnesium, reduced to
80 ± 4 pS with 10 mM internal magnesium) and
sensitivity to voltage and calcium (Fig.
1 A-C). Over the physiological voltage range of AtT20 D16:16 cells, more than 75% (not
shown) of single BK channels are half-maximally activated at positive
(20-30 mV) potentials at "resting" 0.1 µM
intracellular free calcium [Ca2+]i levels (Fig.
1B). At levels of intracellular free calcium observed during
secretagogue stimulation (1 µM) BK channels are maximally
activated (Fig. 1B, half-maximal activation < 40 mV,
not shown).
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Dexamethasone Does Not Modify Single Channel BK Channel Properties or Expression Levels-- Pretreatment of AtT20 D16:16 cells with a maximally effective concentration (1 µM) of the synthetic glucocorticoid agonist, dexamethasone (3, 10), had no significant effect on single channel slope conductance (125 ± 2 and 80 ± 3 pS with 2 and 10 mM internal magnesium, respectively, Fig. 1A) or sensitivity to voltage or calcium in inside-out patches (Fig. 1, A-C). Half-maximal activation of BK channels in 0.1 µM [Ca2+]i in greater than 70% of patches was observed between 20-30 mV (Fig. 1B).
Immunoblotting of crude plasma membrane fractions from control and dexamethasone-treated AtT20 D16:16 corticotropes using an affinity purified antibody (
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Single BK Channels Are Inhibited by Activation of Closely
Associated Protein Kinase A--
In eight of eight control patches
application of cAMP (0.1 mM) to the intracellular face of
the patch in the presence of 1 mM Mg-ATP and 0.5 µM [Ca2+]i resulted in a
significant inhibition of mean channel open probability, Po (expressed
as the percentage of change of pretreatment Po, 72.9 ± 9.5%
p < 0.01 t test, determined 10 min after
cAMP application compared with pretreatment Po, n = 8, Fig. 3, A-C) that was maximal
within 10 min and was maintained for more than 30 min. On washout of
cAMP mean channel open probability gradually returned toward
pretreatment levels; this reversal was accelerated by removal of ATP
from the intracellular face of the channel (not shown). The inhibitory
action of cAMP was mediated through protein kinase A-like activity
closely associated with the channel in the patch because no significant
inhibition of Po was observed on application of cAMP in the presence of
the specific protein kinase A inhibitor peptide, PKI(5-24)
(percentage of change in Po, 0.9 ± 5.3%; n = 4)
or in the absence of ATP (percentage of change in Po, 7.0 ± 10.1%; n = 4) (Fig. 3C).
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Pretreatment of Cells with Dexamethasone Attenuates PKA-mediated
Inhibition of BK Channels in Isolated Inside-Out Patches--
In
parallel experiments, application of cAMP to the intracellular face of
inside-out patches from dexamethasone-treated cells resulted in a
significantly attenuated inhibition of mean channel open probability
compared with inhibition observed in patches from control cells (Fig.
3, B and C). In patches from
dexamethasone-treated cells the percentage of change in Po was
22.4 ± 7.1%, n = 9 (compared with
72.9 ± 9.5%, n = 8, in control patches, p < 0.01 t test, Fig. 3C). The blockade of
PKA-mediated inhibition was not a result of delayed responsiveness to
cAMP (Fig. 3B); in addition the effect of cAMP was mediated
through activation of endogenous PKA (Fig. 4B).
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An Okadaic Acid-sensitive Phosphatase, Closely Associated with BK
Channels, Is Required for Dexamethasone Action--
The thiophosphate
of ATPS can be used by protein kinases to phosphorylate target
proteins, but the resultant phosphoprotein is largely resistant to
dephosphorylation. Using ATP
S as the phosphate donor in place of ATP
in patches from dexamethasone-treated cells, cAMP inhibited Po to the
same extent as that observed in control cells (percentage of change in
Po,
60.8 ± 12.3%, Fig. 3C). These data suggest that
a closely associated protein phosphatase is responsible for the
attenuation of PKA-mediated inhibition of single BK channels in
isolated patches from dexamethasone-treated cells.
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DISCUSSION |
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This study demonstrates that (i) BK channels in AtT20 D16:16 corticotropes are dynamically regulated by protein kinase A and protein phosphatase 2A-like activity closely associated with the BK channel complex and (ii) glucocorticoid regulation of BK channels is dependent upon protein phosphatase 2A activity at the level of the BK channel complex. Importantly, this action of glucocorticoids is context-sensitive because glucocorticoids do not modify the intrinsic properties (calcium or voltage sensitivity) of the BK channel; rather they block PKA-mediated inhibition of BK channel activity. These data support our previous electrophysiological and secretion studies in metabolically intact AtT20 D16:16 cells (3), suggesting that glucocorticoid-induced proteins render BK channels insensitive to inhibition by PKA and that this action of steroids is central to the mechanism of early inhibition of adrenocorticotropin hormone secretion in this system. Furthermore, these data support a growing body of evidence that suggests that reversible phosphorylation of ion channels acts as a dynamic process to finely tune ion channel behavior (12-15, 23).
Mechanism of Glucocorticoid Regulation of BK Channels?-- In order to directly examine the effects of PKA activation and protein phosphatases on BK channel behavior in this paper, we examined regulation in isolated patches of membrane from cells that had been pretreated with a maximally effective dose of glucocorticoid so that the full effects of steroid-induced proteins could be exerted on the channel complex. Because PKA-mediated inhibition of BK channel activity was significantly attenuated in isolated patches of membrane, as we previously observed in whole cell current recordings (3), these data strongly suggest that glucocorticoid-induced proteins exert their effect through pathways that are tightly associated with the BK channel complex. Thus it is reasonable to exclude effects of steroid that require the maintained presence of a diffusible mediator. For example, arachidonic acid metabolites and cGMP exert powerful activation of BK channels in pituitary cells through activation of protein phosphatases (14, 15); however, glucocorticoids inhibit arachidonic acid release (24) and have no effect on cGMP levels in AtT20 D16:16 cells.2
Several lines of evidence suggest that the blockade of PKA-mediated inhibition of BK channel activity by dexamethasone is a result of modified PP2A-like activity closely associated with the BK channel complex. Firstly, cAMP inhibited BK channel activity in patches from dexamethasone-treated cells when endogenous phosphatase activity was blocked by 10 nM calyculin A or okadaic acid. Secondly, using thiophosphate (ATPConclusions and Perspectives-- The data in this report demonstrate that glucocorticoid inhibition of BK channel activity is dependent upon protein phosphatase 2A activity closely associated with the BK channel complex. Such a mechanism may be a common determinant to allow the reciprocal regulation of calcium-activated potassium channels by glucocorticoid-induced proteins and cAMP-dependent protein phosphorylation in excitable cells. Identification of the BK channel complex as a target for glucocorticoid action should allow us to characterize glucocorticoid-induced proteins involved in ion channel regulation and provide further insights into the mechanism and role of rapid glucocorticoid regulation of excitability in neuroendocrine and neuronal cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. F. A. Antoni and Dr. D. L. Armstrong for critical reading of the manuscript and members of the Membrane Biology Group for helpful discussions during this work.
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FOOTNOTES |
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* This work was supported by Wellcome Trust Grants 038763/Z and 046787/Z.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Physiology, The Medical School, Teviot Place, University of Edinburgh, Edinburgh EH8 9AG, Scotland, UK. Tel.: 44-131-650-3253; Fax: 44-131-650-6527; E-mail: Mike.Shipston{at}ed.ac.uk.
1
The abbreviations used are: PKA,
cAMP-dependent protein kinase; BK, large conductance
calcium- and voltage-activated potassium channels; ATPS, adenosine
5'-O-(3-thiotriphosphate); PP2A, protein phosphatase 2A;
PKI(5-24), protein kinase A inhibitor peptide; Po, mean
single channel open probability; DEX, dexamethasone; PBS,
phosphate-buffered saline.
2 M. J. Shipston, unpublished data.
3 L. Tian and M. J. Shipston, unpublished data.
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REFERENCES |
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