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(Received for publication, November 13, 1995; and in revised form, January 19, 1996) From the
Adrenal corticosteroids have well known and profound effects on
neurons and neuroendocrine cells, but the underlying cellular
mechanisms are poorly understood. The present study analyzed membrane
currents and ACTH release in AtT20 mouse pituitary corticotrope tumor
cells. Patch-clamp analysis revealed a significant and selective
inhibition of calcium-activated (BK-type) potassium channels upon
activation of protein kinase A by corticotropin-releasing factor or
8-chlorophenylthio-cAMP. The synthetic glucocorticoid dexamethasone had
no effect on potassium currents evoked by depolarization but prevented
the inhibitory effect of protein kinase A activators. The action of
dexamethasone had the hallmarks of protein induction, i.e. a
lag time and sensitivity to inhibitors of DNA transcription and mRNA
translation. In parallel, the specific BK channel blocker iberiotoxin
abolished early glucocorticoid inhibition of corticotropin-releasing
factor-stimulated ACTH secretion. In summary, the present data show
that glucocorticoid-induced proteins render BK-type channels resistant
to inhibition by protein kinase A and that this action of the steroid
is pivotal for its early inhibitory effect on the secretion of ACTH.
Stressors provoke a cohort of homeostatic defense mechanisms by
the central nervous, the immune, and the metabolic control systems of
the body(1) . Adrenal corticosteroids released during the
stress response terminate many of these adaptive responses through the
rapid induction of proteins, the nature and mechanism of which are not
understood(1, 2, 3, 4) . A common
motif of corticosteroid action in hippocampal neurons(5, 6) and pituitary somatolactotrope cells (7) is the
regulation of potassium channels that are important determinants of
cellular excitability. In hippocampal neurons, the target of
glucocorticoid action has not been defined. In somatolactotrope cells,
glucocorticoid induction of Kv1.5 voltage-regulated potassium channel
subunits has been demonstrated (7) . A physiologically
important early action of glucocorticoids is the suppression of
CRF( In endocrine
cells and neurons large conductance calcium- and voltage-activated
potassium (BK) channels have been proposed as important negative
feedback regulators of voltage-dependent calcium influx(18) .
Such channels have been previously identified in AtT20 corticotrope
tumor cells(19, 20) . This report describes a
correlated study of the regulation of BK channels and ACTH release by
CRF and glucocorticoids in the mouse anterior pituitary corticotrope
cell line AtT20. The data demonstrate that glucocorticoid-induced
proteins prevent protein kinase A-dependent inhibition of BK channels
and that this is pivotal to early glucocorticoid inhibition of
CRF-induced ACTH secretion.
For determination of outward potassium currents cells
were voltage-clamped at -60 mV in physiological saline containing
(in mM): 140 NaCl, 5 KCl, 25 HEPES, 0.8 MgCl For isolation of high threshold
voltage-activated calcium currents, cells were voltage-clamped at
-40 mV in (mM): 120 N-methyl-D-glucamine, 30 TEA-Cl, 2 MgCl
In control cells voltage clamped through
amphotericin-perforated patches in physiological saline at -60
mV, a 100-ms depolarization evoked large outward currents that reached
a steady-state level before the end of the pulse. Variations in the
rate of inactivation of the outward currents were observed between
cells (compare Fig. 1A and Fig. 2B)
that were independent of passage number or incubation temperature
(19-24 °C). This slow inactivation was observed in
Figure 1:
CRF inhibits the BK component of the
outward steady-state current via activation of protein kinase A in
intact AtT20 D16:16 cells. A, representative (1 of 7 cells)
leak-subtracted traces of voltage-activated outward current (at
+30 mV) in control cells before and after exposure to 100 nM CRF. AtT20 cells were voltage-clamped through amphotericin
B-perforated patches at -60 mV in physiological saline containing
1 µM TTX. B, the current/voltage relationship
determined from the same cell in A. Average steady-state
outward current was determined between 90 and 100 ms at each potential
as described under ``Materials and Methods.'' C,
time course of 100 nM CRF inhibition of outward steady-state
potassium current (filled circle, measured at +30 mV) and
CRF-stimulated intracellular cAMP accumulation (open circle, n = 3). Inhibition of outward current is expressed as
the percentage inhibition of the outward steady-state current at each
time point with respect to the pre-CRF-treated current amplitude (Io). Intracellular cAMP accumulation was determined under
identical conditions, in the absence of phosphodiesterase inhibitors,
as described under ``Materials and Methods.'' D,
pretreatment of cells with the protein kinase A inhibitor, Rp-cAMPS
(100 µM), or the selective BK inhibitor, IbTx (100
nM), blocks CRF inhibition of outward steady-state current.
Data are expressed as the percent change in the control outward
steady-state current, Io (at +30 mV) by CRF (100
nM) alone, Rp-cAMPS (100 µM) + CRF, IbTx
(100 nM) alone, and IbTx + CRF. Number in parentheses represents number of cells in each group. Means
± S.E. are shown; *, p < 0.05 (non-parametric
Kruskal-Wallis test). E, representative records (1 of 4) from
a cell-attached patch in 140 mM KCl during consecutive (0.1
Hz) 100-ms depolarizations to +30 mV before (control) and 5 min
after CRF application. Open (o) and closed (c) states
of channel are shown. F, representative plot of BK channel
mean open probability versus time from a cell-attached patch
recording as in E. Bath application of 100 nM CRF
reduces average open probability (Po) of the
Figure 2:
Dexamethasone prevents protein kinase
A-mediated inhibition of BK currents in intact AtT20 D16:16 cells. A, representative (1 of 8 cells) leak-subtracted traces of
voltage-activated outward current (at +30 mV) before and after
exposure to 8-CPT-cAMP (0.1 mM, 10 min) in AtT20 cells
pretreated for 2 h with the glucocorticoid agonist, dexamethasone (1
µM). B, 8-CPT-cAMP inhibits outward steady-state
current in dexamethasone-treated cells pretreated with the mRNA
transcription inhibitor, actinomycin D (0.1 mM applied 15 min
before and during dexamethasone treatment). A representative trace at
+30 mV from 1 of 3 cells is shown. C, current/voltage
relationship of steady-state outward current density from control (open squares, n = 43) and
dexamethasone-treated (filled squares, n = 29)
cells. Outward steady-state current at each potential was normalized to
membrane capacitance to compensate for variations in cell size and
expressed as mean current density, pA/pF. D, summary of effect
of bath application of CRF (100 nM, 5 min), 8-CPT-cAMP (5 min,
0.1 mM), IbTx (20 min, 100 nM), or TEA (2 min, 1
mM) on outward steady-state potassium current determined at
+30 mV. Data are expressed as the percentage change (% change
Io) in outward steady-state current compared with pretreated
control current amplitude (I
These data show that the reduction of outward
potassium current by CRF is through activation of protein kinase A.
This finding corroborates previous evidence that protein kinase A
activation in corticotropes results in membrane depolarization and
sustained calcium influx through voltage-sensitive L-type calcium
channels(19, 23, 24) ; in parallel,
inhibition of protein kinase A by various methods also blocks the
hormone secretory response to CRF in AtT20
cells(25, 26) .
Bath application of 100 nM CRF significantly (p < 0.05) reduced the average open probability of single large
conductance ( These results clearly
show that CRF and cAMP suppress the activity of BK channels in AtT20
cells under conditions when these channels are exposed to elevated
intracellular free calcium levels that enhance their open
probability(20) . As BK channels are thought to act as
immediate negative feedback inhibitors of voltage-dependent calcium
influx, inhibition of these channels is a key element for optimal
activation of calcium channels involved in hormone secretion (18) . Previous evidence shows that BK channels may be up-
or down-regulated by reversible cAMP-dependent
phosphorylation(28) . However, in pituitary cells only protein
kinase A-dependent inhibition has been found so
far(29, 30) , and the present study is the first
example of inhibition of BK channels by a physiologically relevant
cAMP-mobilizing hypothalamic peptide, CRF.
In the absence of protein kinase A
activation, dexamethasone had no significant effect on mean current
density, threshold of activation, or sensitivity to 100 nM IbTx or 1 mM TEA of the steady-state outward current (Fig. 2, C and D), in agreement with a
previous study(16) . Similar to the findings with the
macroscopic current, no significant inhibition of single BK channels
was observed by CRF in cell-attached patch recordings (0/5 patches)
from dexamethasone-treated cells. These observations show that
dexamethasone action is mediated by newly induced proteins and is
selective for BK channels inhibited by protein kinase A.
Glucocorticoids have also been reported to enhance a 4-aminopyridine
(4-AP)-sensitive potassium current in AtT20 cells(16) . In
functional secretion assays 4-AP affects ACTH release; however, 4-AP
alone does not reverse glucocorticoid inhibition of CRF-stimulated ACTH
release(16) . In contrast, blockade of BK channels alone with
IbTx (Fig. 3) completely reverses the inhibitory effect of
dexamethasone suggesting BK channels are a primary target for
glucocorticoid action in AtT20 cells.
Figure 3:
IbTx
prevents early glucocorticoid inhibition of CRF-stimulated ACTH
secretion. A, static incubation assays of CRF-stimulated ACTH
secretion were performed at room temperature (24 °C) under the
conditions used to monitor outward currents as described under
``Materials and Methods.'' CRF (100 nM) was applied
for 30 min. TEA (1 mM) was applied during the CRF exposure,
and IbTx (100 nM) was applied >15 min before and during CRF
exposure. Cells were treated for 2 h with 1 µM dexamethasone or vehicle (0.01% Me
The early inhibitory effect of
glucocorticoids on CRF-stimulated ACTH release also requires mRNA and
protein synthesis(2, 8, 10, 14) ,
and in this respect the characteristics of the action of dexamethasone
on BK channels and ACTH release are identical. The selectivity of the
effect of dexamethasone on protein kinase A-inhibited BK channels also
helps to explain why early glucocorticoid inhibition suppresses
CRF-stimulated ACTH release but does not affect basal output of hormone (2, 8, 13, 14) .
As shown by others previously (12) CRF enhanced peak pharmacologically isolated high
voltage-activated calcium currents in control (mean increase at
+20 mV was 44 ± 15%, n = 4) and
dexamethasone-treated (33 ± 5%, n = 5) cells.
Similar enhancement was also seen with 8-CPT-cAMP (n =
2). Dexamethasone pretreatment (2 h, 1 µM) had no
significant effect on peak calcium current density (measured at
+20 mV: control, 8.9 ± 1.5 pA/pF (n = 8);
dexamethasone, 9.2 ± 1.4 pA/pF (n = 7). On
the basis of these data it seems reasonable to suggest that direct
modulation of calcium influx through high voltage-activated calcium
channels is not responsible for the effects of dexamethasone on BK
currents. Moreover, previous work indicates a reduction (11, 32) or no significant change (10) in the
average levels of intracellular free calcium after dexamethasone
treatment in AtT20 cells, thus excluding increases in intracellular
free calcium levels derived from intracellular sources as mediators of
the effect of the steroid.
The IC
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9197-9200
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)-induced ACTH secretion from anterior pituitary
corticotropes (for reviews see (8) and (9) ). CRF
stimulates ACTH release through activation of protein kinase A and the
enhancement of calcium influx through voltage-operated calcium
channels(10, 11, 12) . In turn,
glucocorticoids rapidly (within 2 h) inhibit CRF-stimulated ACTH
secretion through the induction of new
protein(s)(2, 8, 10, 13, 14) ,
while basal secretion remains unchanged. The early inhibition of
stimulated ACTH release in AtT20 corticotropes is antagonized by
membrane depolarization(13, 15) , calcium channel
activator drugs(11, 16) , and potassium channel
blockers(15, 16) , collectively suggesting that the
actions of glucocorticoids may involve potassium channels. However, the
well established glucocorticoid induction of the Kv1.5 channel protein
does not occur in AtT20 corticotropes(17) .
AtT20 D16:16 Cell Culture
Clonal mouse anterior
pituitary (AtT20 D16:16, passage 19-30) cells were maintained as
described previously (14) except that cells were passaged by
brief trypsinization 1 week before replating on glass coverslips. Cells
(3-7 days postplating) were treated with dexamethasone or vehicle
(0.01% Me
SO) for 2 h at 37 °C in serum-free
HEPES-buffered Dulbecco's modified Eagle's medium. Cells
were then transferred to physiological saline containing (in
mM): 140 NaCl, 5 KCl, 25 HEPES, 0.8 MgCl, 2
CaCl, 30 glucose, and 0.001 tetrodotoxin (TTX), pH 7.4, at
room temperature (19-24 °C) for electrophysiological
recording. Regulation of currents in control and dexamethasone-treated
cells was performed in parallel on the same passage of cells to avoid
intrapassage variations.Electrophysiology
Whole cell currents were
recorded under voltage clamp using the amphotericin B (200 µg/ml)
perforated patch configuration of the whole cell patch clamp recording
technique(21) . Data acquisition and voltage protocols were
controlled by an Axopatch 200 amplifier and pCLAMP software (Axon
Instruments Inc., Foster City, CA). All traces are leak-subtracted
records from cells with compensated series resistance of <15
megaohms. Pipettes were manufactured from Garner 7052 glass with
resistances of 1.5-3 megaohms in physiological saline after fire
polishing., 2
CaCl, 30 glucose, and 0.001 TTX, pH 7.4. The patch pipette
contained (in mM): 95 KCH
SO, 55 KCl,
10 HEPES, 2 MgCl, 0.1 CaCl, and 200 µg/ml
amphotericin B, pH 7.35. Outward potassium currents were evoked by
100-ms step depolarization (-30 to +50 mV), and the average
steady-state current amplitude between 90 and 100 ms was determined at
each potential. Outward currents were stable for >1 h in this
configuration. Single BK channel events were recorded using the
cell-attached patch configuration. Cells were voltage-clamped at 0 mV
in high potassium saline to eliminate the membrane potential, and the
magnesium to calcium ratio adjusted to limit calcium entry (in
mM: 140 KCl, 5 NaCl, 5 MgCl, 10 HEPES, 1
CaCl, 30 glucose, pH 7.4. The patch pipette contained
physiological saline supplemented with 0.001 mM TTX and 100
nM apamin, and single channel events were recorded during
repeated (0.1 Hz) 100-ms depolarizations to +30 mV. Under these
conditions control P
values at +30 mV were
similar (<0.5) to that recorded in isolated patches depolarized to
+30 mV and exposed to <200 nM intracellular free
calcium(20) . (),
20 HEPES, 10 CaCl, and 20 glucose, pH 7.35, with
CHSOH. The patch pipette contained (in
mM): 95 CsCHSO, 35 CsCl, 5
MgCl, 40 HEPES, and 200 µg/ml amphotericin B, pH 7.35,
with CsOH. Calcium currents were evoked by 100-ms depolarization, and
peak calcium current was determined. Under the recording conditions
used currents were stable for >30 min, and inward calcium current
was completely blocked by 200 µM Cd
and
70-80% by 1 µM nifedipine.ACTH Secretion and Intracellular cAMP
Determination
For intracellular cAMP determination and ACTH
secretion studies, cells were plated (5 
10
cells/well) in 24-well plates and used 4-5 days
postplating. Cells were incubated in HEPES-buffered Dulbecco's
modified Eagle's medium at 37 °C for 2 h with 1 µM dexamethasone or vehicle (0.01% MeSO). Cells were
washed twice and equilibrated for 15 min at room temperature in
physiological saline used for outward potassium current determination
except that TTX was excluded and 0.1% w/v bovine serum albumin was
included to aid ACTH recovery. CRF (100 nM) was then applied
for various times as indicated in the legends. IbTx was applied to the
cells 15 min before CRF application. For intracellular cAMP
determination, medium was aspirated and cells lysed in ice-cold 0.1 N HCl by freeze thawing and cAMP content in the acid extracts
determined. ACTH and cAMP were assayed using specific double
precipitation radioimmunoassays as described previously(14) .Reagents
CRF and ACTH were from Bachem (UK) Ltd.,
Saffron Walden, UK; 8-CPT-cAMP and Rp-cAMPS were from Boehringer
Mannheim UK, Lewes, East Sussex, UK; iberiotoxin was from the Peptide
Institute, Japan. All other reagents were from Sigma or Aldrich.
Dexamethasone was stored at -20 °C at 10 mM in
MeSO. Final vehicle concentrations were <0.01% and had
no effect on calcium or potassium currents.
20%
of both control and glucocorticoid-treated cells. No qualitative
differences in regulation of steady-state current, measured between 90
and 100 ms, reported here were observed between inactivating and
non-inactivating currents.
120-picosiemens BK channel in cell-attached patches. P was determined during consecutive (0.1 Hz)
100-ms patch depolarization to +30 mV and plotted as a function of
time.
) as described in Fig. 1C. Cells were voltage-clamped at -60 mV as
described in Fig. 1and under ``Materials and
Methods.'' The number in parentheses indicates
number of cells per group. Means ± S.E. are
shown.
CRF Inhibition of Potassium Currents Is Mediated by Protein
Kinase A
Bath application of a CRF concentration (100
nM) maximally effective with respect to ACTH release in this
system (14) inhibited the steady-state outward potassium
current (mean ± S.E., 29.6 ± 8.6%, n = 7, p < 0.01, Kruskal-Wallis test, at +30 mV) at all
potentials examined (Fig. 1, A and B). The
time course of CRF inhibition of potassium currents followed the time
course of CRF-stimulated intracellular cAMP accumulation (Fig. 1C). The effect of CRF was significantly reduced
by pretreatment of cells with the protein kinase A inhibitor Rp-cAMPS (Fig. 1D)(22) . Steady-state outward potassium
current was also significantly inhibited (30.3 ± 2.3%, n = 10, at +30 mV, p < 0.01) after bath
application of the cell-permeant protein kinase A activator, 8-CPT-cAMP
(0.1 mM).The Target of Protein Kinase A Action Is the BK Potassium
Channel
Both iberiotoxin (100 nM), a highly specific
blocker of BK type channels (27) and TEA (1 mM), a
broader spectrum blocker, inhibited the steady-state outward current by
34.0 ± 6.5% (n = 4, Fig. 1D) and
52.9 ± 6.3% (n = 9), respectively. No
significant inhibition of the residual outward current by CRF (or
8-CPT-cAMP) occurred in cells pretreated with 100 nM iberiotoxin (mean inhibition, 6.2 ± 5.6%, n = 4, Fig. 1D) or 1 mM TEA (mean
inhibition, 5.1 ± 7.2%, n = 4). Similar results
were obtained with 100 nM charybdotoxin (n =
2).120 picosiemens) BK channels in 4/6 patches in
cell-attached patch recordings (Fig. 1, E and F). The time of onset (2 min) of the effect of CRF was
identical to that seen for the macroscopic current (compare Fig. 1, C and F).Dexamethasone Prevents Inhibition of Potassium Current by
Protein Kinase A
Pretreatment with 1 µM dexamethasone for 2 h, which produces maximal inhibition of
CRF-stimulated ACTH release (14) , blocked the inhibition of
outward steady-state potassium current by 8-CPT-cAMP (Fig. 2, A and D) or CRF (Fig. 2D). This
effect of dexamethasone was prevented with actinomycin D (0.1
mM, Fig. 2B) or puromycin (2 µM, n = 2), indicating a requirement for de novo RNA and protein synthesis, a defining feature of early
inhibition(8, 14) . In addition an acute (10 min)
exposure to dexamethasone did not block CRF inhibition of the
steady-state current (not shown).
SO) as appropriate.
ACTH release for each treatment in dexamethasone-treated cells (open box) is expressed as the percent of the respective
control (absence of dexamethasone, filled box) stimulus
(100%). In these studies CRF (100 nM) stimulated ACTH release
to 157 ± 6% (n = 6) of basal levels. IbTx (100
nM) or TEA (1 mM) alone elicited a small but
significant (p < 0.05) ACTH response (114 ± 2%, n = 3 and 127 ± 7%, n = 5 of
basal, respectively). IbTx had no significant effect on CRF-stimulated
ACTH secretion (155 ± 7.9%, n = 6 of basal
release). TEA (1 mM) significantly (p < 0.05,
non-parametric Kruskal-Wallis test) enhanced CRF-stimulated ACTH
release to 185 ± 10.5%, (n = 5) of basal. Means
± S.E. are shown; *, p < 0.01 compared with control
ACTH release (non-parametric Kruskal-Wallis
test).
Effects of Dexamethasone Are Not Secondary to Changes in
Calcium Currents
As intracellular calcium ions are potent
activators of BK channels and glucocorticoids reportedly (31) enhance calcium entry to stimulate potassium currents in
hippocampal neurons, the actions of CRF and dexamethasone on calcium
currents were also analyzed.Iberiotoxin-sensitive Channels Are Pivotal for Early
Glucocorticoid Inhibition of CRF-stimulated ACTH Release
Under
the same conditions that were used to monitor outward potassium
currents, CRF (100 nM) stimulated ACTH release to 157 ±
6% (n = 6) of basal levels. IbTx (100 nM) or
TEA (1 mM) alone elicited a small but statistically
significant (p < 0.05 non-parametric Kruskal-Wallis test)
ACTH response (114 ± 2%, n = 3 and 127 ±
7%, n = 5 of basal, respectively). IbTx had no
significant effect on CRF-stimulated ACTH secretion (155 ± 7.9%, n = 6 of basal release). TEA (1 mM)
significantly (p < 0.05, Kruskal-Wallis test) enhanced
CRF-stimulated ACTH release to 185 ± 10.5% (n =
5) of basal. AtT20 cells contain multiple voltage-activated potassium
conductances(16) , and in this system 1 mM TEA blocks
a greater proportion of outward, steady-state, voltage-activated
current than 100 nM IbTx (Fig. 2D). The
effects of these inhibitors are not additive (not shown), indicating
that TEA blocks iberiotoxin-sensitive BK channels as well as other
potassium conductances in this system.
of
dexamethasone to block CRF-induced ACTH release was 7.7 ± 1.9
nM (n = 6). Importantly, IbTx (100
nM) as well as TEA (1 mM) completely blocked the
inhibitory effect of 1 µM dexamethasone on CRF-stimulated
ACTH secretion (Fig. 3). Furthermore, dexamethasone (1
µM) had no significant inhibitory effect on ACTH secretion
stimulated by IbTx or TEA alone.Final Comment
Taken together with previous
results(8, 12) the present data suggest that
concerted cAMP-dependent phosphorylations of L-type calcium channels as
well as BK-type potassium channels are required for triggering the ACTH
secretory response to CRF. Inhibition of BK channels by cAMP-dependent
phosphorylation prevents the calcium-induced feedback hyperpolarization
mediated by these channels and thus enhances voltage-activated calcium
entry(18) . The critical role of this process in CRF-stimulated
ACTH secretion is shown by the observation that dexamethasone prevents
protein kinase A inhibition of BK channels and in turn that selective
blockage of BK channels abolished the early inhibition of ACTH release.
)SO, dimethyl sulfoxide; TTX, tetrodotoxin; IbTx,
iberiotoxin; BK, large conductance calcium- and voltage-activated
potassium channel; TEA, tetraethylammonium; 8-CPT-cAMP,
8-chlorophenylthio-cAMP; Rp-cAMPS, adenosine 3`,5`-cyclic
monophosphothioate-Rp; 4-AP, 4-aminopyridine; pF, picofarads.)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. J. Shipston, R. R. Duncan, A. G. Clark, F. A. Antoni, and L. Tian Molecular Components of Large Conductance Calcium-Activated Potassium (BK) Channels in Mouse Pituitary Corticotropes Mol. Endocrinol., October 1, 1999; 13(10): 1728 - 1737. [Abstract] [Full Text]
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L. Tian, J. A C Philp, and M. J Shipston Glucocorticoid block of protein kinase C signalling in mouse pituitary corticotroph AtT20 D16:16 cells J. Physiol., May 1, 1999; 516(3): 757 - 768. [Abstract] [Full Text] [PDF]
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A R Evans, M R Vasko, and G D Nicol The cAMP transduction cascade mediates the PGE2-induced inhibition of potassium currents in rat sensory neurones J. Physiol., April 1, 1999; 516(1): 163 - 178. [Abstract] [Full Text] [PDF]
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L. Tian, H.-G. Knaus, and M. J. Shipston Glucocorticoid Regulation of Calcium-activated Potassium Channels Mediated by Serine/Threonine Protein Phosphatase J. Biol. Chem., May 29, 1998; 273(22): 13531 - 13536. [Abstract] [Full Text] [PDF]
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L. Vergara, E. Rojas, and S. S. Stojilkovic A Novel Calcium-Activated Apamin-Insensitive Potassium Current in Pituitary Gonadotrophs Endocrinology, July 1, 1997; 138(7): 2658 - 2664. [Abstract] [Full Text] [PDF]
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L. Tian, R. R. Duncan, M. S. L. Hammond, L. S. Coghill, H. Wen, R. Rusinova, A. G. Clark, I. B. Levitan, and M. J. Shipston Alternative Splicing Switches Potassium Channel Sensitivity to Protein Phosphorylation J. Biol. Chem., March 9, 2001; 276(11): 7717 - 7720. [Abstract] [Full Text] [PDF]
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