(Received for publication, July 7, 1994; and in revised form, December 21, 1994)
From the
Voltage-gated K channels play an essential role
in the production of action potential activity by excitable cells.
Recent studies have suggested that expression of K
channel genes may be regulated by stimuli that affect electrical
activity. Elevating the concentration of extracellular KCl causes
membrane depolarization and, thus, is widely used for studying
electrical activity-dependent changes in neurons, muscle, and endocrine
cells. Here we show that elevated KCl decreases Kv1.5 K
channel mRNA expression in clonal pituitary cells without
affecting Kv1.4 and Kv2.1 mRNA levels. K
channel
blockers, which cause depolarization, also produce down-regulation of
Kv1.5 mRNA, while NaCl addition had no effect. Thus, the effect of KCl
is mediated by K
-induced membrane depolarization.
Unlike many known effects of K
, down-regulation of
Kv1.5 mRNA does not require Ca
or Na
influx, or Na
-H
exchange.
Furthermore, the decrease in Kv1.5 mRNA expression is due to inhibition
of channel gene transcription and persists after inhibition of protein
synthesis, excluding a role for induction of intermediary regulatory
proteins. Finally, immunoblots with antibody specific for the Kv1.5
polypeptide show that depolarization for 8 h reduces the expression of
Kv1.5 channel protein. The decrease in K
channel
protein expression caused by depolarization-induced
Ca
-independent inhibition of Kv1.5 gene transcription
may produce a long-term enhancement of pituitary cell excitability and
secretory activity.
K channels control action potential
repolarization and frequency. Recent molecular genetic studies have
shown that the voltage-gated K
channels are multimeric
proteins that are encoded by a large number of genes(1) . This
great genetic diversity allows the production of a large assortment of
K
channels that differ in their functional properties.
Thus, heterogeneity of cell excitability may be increased because of
the large repertoire of K
channel genes expressed in
neurons, muscle, and endocrine cells.
Another potential advantage of
genetic diversity is that long-term changes in excitability could be
produced by differential regulation of K channel gene
expression. We recently reported that glucocorticoids up-regulate Kv1.5
K
channel mRNA and protein in pituitary and cardiac
cells without altering Kv1.4 channel
expression(2, 3, 4, 14) . Expression
of Kv1.4 and Kv1.5 mRNAs is also differentially controlled by a
neuropeptide, protein kinases, and cardiac
hypertrophy(5, 6, 7, 8) . Expression
of K
channel mRNAs also vary during development of the
nervous system and the heart (9, 10, 11, 12, 13) .
Interestingly, the effects of steroid hormones, KCl, and cyclic AMP on
K
channel mRNA expression differ between
tissues(6, 14, 26) . Thus, expression of
K
channel mRNAs is differentially controlled in a
cell-type specific manner. The regulation of K
channel
mRNA levels may have important physiological and pharmacological
consequences. We have found that rapid steroid induction of Kv1.5
K
channel mRNA expression leads to an alteration in
channel protein expression that correlates with changes in
voltage-gated K
current in clonal pituitary
cells(4) . Since glucocorticoids up-regulate Kv1.5 mRNA
expression in the pituitary and heart in
vivo(3, 14) , it is likely that altering channel
gene expression affects cell excitability under physiological
conditions. It has also been found that some heterologously expressed
K
channels are sensitive to clinically important
drugs. For example, Kv1.5 channels are sensitive to the antiarrhythmics
quinidine, verapamil, tedisamil, and to a proarrhythmic
antihistamine(15, 16, 17, 18) .
Therefore, it is possible that altering the expression of Kv1.5
channels affects the actions of these agents. Thus, specific control of
K
channel gene expression might be an important
fundamental mechanism for long-term modulation of excitability that
also alters the efficacy of therapeutic drugs.
Interestingly, recent
studies have raised the possibility that electrical activity might
affect K channel gene expression. For example, it was
shown that drug-induced seizures, which are associated with abnormally
enhanced action potential activity, can decrease expression of two
K
channel mRNAs in the hippocampus(40) .
Furthermore, it has been found that bath application of elevated KCl, a
depolarizing stimulus, alters K
channel mRNA
expression in atrial cardiac cells and clonal pituitary
cells(5, 6, 19) . To date, the effects of
these stimuli on channel transcription and protein expression have not
been examined. Moreover, it is possible that the changes induced by
seizures and KCl might be independent of their effects on membrane
potential. Thus, a role for membrane potential in the control of
K
channel gene expression was not established by these
studies.
Therefore, we chose to study the action of KCl in GH clonal pituitary cells. These cells express a variety of
identified voltage-gated ion channel genes (3, 22) and
show glucocorticoid regulation of Kv1.5 gene expression found in normal
pituitary cells in vitro and in
vivo(2, 3, 4) . Hence, they constitute a
useful model system for studying regulation of K
channel gene expression. Furthermore, we and others have found
that KCl addition down-regulates Kv1.5 mRNA in these
cells(6, 19) . We were intrigued by the fact that
extracellular K
is thought to promote gating activity
by binding to the outer mouth of these
channels(41, 42) . Elevating extracellular
K
also depolarizes the cell membrane and hence
indirectly activates voltage-gated K
channels. Thus,
elevating extracellular K
is in many ways analogous to
applying an agonist to a receptor. Activation of
-adrenergic and
thyrotropin releasing hormone receptors leads to a destabilization of
receptor mRNA(20, 21) . To date, no involvement of
calcium has been demonstrated for those effects. On the other hand, it
has been proposed that KCl induces changes in the synthesis of mRNAs
encoding the c-Fos protein, nicotinic acetylcholine receptors, and
neuropeptides by triggering by depolarization-activated Ca
influx through voltage-gated Ca
channels(34, 35, 36, 37) .
Indeed, it has been hypothesized recently that this mechanism is
involved in KCl regulation of expression of Kv1.4 and Kv1.5
K
channel mRNAs(5, 6) . Hence, we set
out to determine the mechanism and consequence of KCl-induced
down-regulation of Kv1.5 mRNA in clonal pituitary cells.
Here we
report that KCl-induced down-regulation of Kv1.5 mRNA in GH cells is specific and is caused by membrane depolarization
(rather than by the KCl itself). However, it is independent of
Ca
influx and induction of immediate early gene (IEG) (
)expression. We also demonstrate that this effect is due to
inhibition of channel gene transcription. Finally, we establish that
Kv1.5 protein expression is also down-regulated by depolarization.
Depolarization-induced suppression of Kv1.5 gene transcription leading
to decreased channel protein expression may produce a long-term
increase in pituitary cell excitability and secretory activity.
GH pituitary tumor cells were purchased from the
American Type Culture Collection (Rockville, MD) and were grown in
Ham's F-10 medium supplemented with 15% horse serum and 2.5%
fetal bovine serum in 5% CO
at 37 °C in Corning tissue
culture flasks or dishes. In most experiments, cells were treated by
adding the reagent of interest (e.g. an aliquot of 2 M KCl) or vehicle alone to the tissue culture solution. Addition of
calcium chelators caused cells to detach from the plastic dishes.
Therefore, cells were grown on polylysine-coated dishes, or detached
cells were collected by centrifugation. For Na
substitution experiments, the cell culture solution was replaced
with a solution containing 140 mM NaCl or N-methylglucamine HCl, 5.4 mM KCl, 1.8 mM CaCl
, 0.8 mM MgCl
, 5.4 mM KCl, 10 mM Hepes (pH 7.5).
RNA was extracted from
cultured cells by a single step guanidinium
thiocyanate-phenol-chloroform procedure(23) . Yield of total
RNA was determined based on the measured A. For
Northern blots, aliquots of 5 µg of RNA were electrophoresed in
1.0-1.2% agarose-formaldehyde gels and were transferred to Nytran
membranes (Schleicher and Schuell) by capillary blotting.
Prehybridizations and hybridizations using the rat Kv1.5 cDNA probes
described by Swanson et al.(11) (who originally named
the gene Kv1) were carried out as described in (3) . A fragment
of Kv1.4 DNA was generated by digesting clone RK3 (24) with XhoI and BglII. A fragment of Kv2.1 DNA was generated
by digesting a fragment of the drk1 gene (25) with HindIII and BamHI to yield nucleotides 359-1747 of
the gene. Purified cDNA fragments were labeled with
[
-
P]dCTP (3000 Ci/mmol; DuPont NEN Research
Products) to 5-10
10
cpm/µg with a
Boehringer Mannheim random primer kit. Labeled probe was added to the
hybridization solutions to 3
10
cpm/ml.
Hybridization signals were quantitated by densitometric scanning of
autoradiograms. Normalization for differences in loading was done by
densitometry of the 28 S ribosomal RNA ethidium bromide signal (27) or, in some cases, rehybridizing blots with probes to
either rat cyclophilin (generously provided by Dr. James Douglass) or
-actin. These normalization methods yielded similar results.
For isolation of nuclei, cells grown on two 100-mm dishes were
washed with ice-cold Tris-buffered saline and collected in 1 ml of
lysis buffer (10 mM Tris-HCl (pH 8.0), 5 mM MgCl, 100 mM KCl, 1 mM EDTA, 2
mM dithiothreitol, and 0.05% Triton X-100). The cell
suspension was kept on ice for 5 min and centrifuged at 2,000
g for 1 min. The pelleted nuclear fractions were then washed
with 1 ml of the same buffer and stored in 200 µl of nuclear stock
solution (HEPES-KOH (pH 7.5), 5 mM MgCl
, 0.1
mM EDTA, and 50% glycerol) at -80 °C until use. The
number of nuclei in this preparation were approximately 5
10
. Nuclear transcription assay was performed, and RNAs
were purified as described previously(4) . All cDNAs used for
hybridization were constructed into pGEM1 (Promega). Plasmid DNAs were
linearized at unique restriction enzyme sites in the polylinker region
and denatured in 0.1 M NaOH at 37 °C for 30 min. The
denatured DNA was applied to a Nytran membrane (DuPont NEN) (2
µg/slot) in a slot-blot apparatus (Life Technologies, Inc.).
Hybridization and washing of the membrane were performed under the same
conditions as described previously(4) .
For immunoblotting,
cells grown on 60-mm dishes were washed with ice-cold Tris-buffered
saline containing 1 mM EDTA and harvested in the same buffer.
The cells were then homogenized in 100 µl of homogenizing buffer
(20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% Triton X-100)
by pipetting. The cell suspension was centrifuged at 3,000 g for 5 min to remove nuclear debris. Protein concentration
was determined using Bio-Rad protein assay solution with human
immunoglobulin as a standard. The proteins were separated on an 8%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
The membrane was probed with polyclonal antibody specific for Kv1.5
polypeptide(4) , and an immunoreactive protein was detected by
the ECL chemiluminescence method (Amersham).
Densitometry measurements from Northern and immunoblot experiments were normalized to the control value. Statistical significance for pairwise comparisons to control was calculated with a one-sample t test using two-tailed p values unless otherwise stated. For multiple comparisons, the Bonferroni test was used. Results with p values of <0.05 were accepted as statistically significant. Error bars in the figures show standard error of the mean.
Figure 1:
Addition of 50 mM KCl
specifically down-regulates Kv1.5 mRNA in GH cells. A, autoradiograms of the three K
channel
mRNAs are shown with arrows on the left indicating
the bands quantitated by densitometry. The quantitated transcript sizes
are approximately 3.5, 6, and 11 kilobases for Kv1.5, Kv1.4, and Kv2.1,
respectively. B, open circles, triangles,
and closed circles represent mRNA levels for Kv1.5, Kv1.4, and
Kv2.1, respectively. n
3 for all points.** indicate p < 0.01. * indicates p <
0.05.
Since KCl was added hypertonically, osmolarity and Cl concentration were altered. To address the importance of these
changes, we compared the actions of the addition of KCl and NaCl.
Hypertonic addition of 5-50 mM NaCl did not
significantly affect Kv1.5 mRNA level (Fig. 2). In contrast,
addition of KCl decreased Kv1.5 mRNA expression is a dose-dependent
manner. Even with the addition of only 5 mM salt, KCl produced
a significantly lower steady state concentration of Kv1.5 mRNA than
NaCl (p < 0.05). A role for osmolarity changes was also
excluded by the finding that isoosmotic addition of KCl also
down-regulated Kv1.5 mRNA (n = 3). Hence, inhibition of
Kv1.5 mRNA expression is caused by addition of K
rather than by increasing the concentration of Cl
or osmolarity.
Figure 2: Dose-response relationships for down-regulation of Kv1.5 mRNA by KCl and NaCl. Varying amounts of each salt were added to the medium, and cells were treated for 3 h. n = 3.
Addition of K could act by
depolarizing the cell membrane. If this were the case, then Kv1.5 mRNA
should be down-regulated by another depolarizing stimulus. Therefore,
we tested the effect of applying the K
channel
blockers tetraethylammonium chloride (TEA-Cl) and 4-aminopyridine
(4-AP). Perforated patch clamp measurements showed that acute
application of 50 mM TEA with 1 mM 4-AP depolarized
GH
cells. However, unlike elevating extracellular
K
, the effect partially reversed within 5 min, but
then was sustained (data not shown). This partial reversal was likely
due to inactivation of Ca
channels and activation of
Ca
-activated chloride channels, both of which would
tend to repolarize the membrane potential. These electrophysiological
data suggest that if K
acts via depolarization, then
TEA and 4-AP ought to down-regulate Kv1.5 mRNA, albeit to a lesser
degree than 50 mM KCl. We found that 3-h applications of 50
mM TEA with 1 mM 4-AP decreased Kv1.5 mRNA by 29
± 6% (n = 5, p < 0.01). Thus, the
K
-induced down-regulation of Kv1.5 mRNA appears to be
mediated by membrane depolarization.
Figure 3:
Dihydropyridine Ca
channel inhibitors do not block the effect of depolarization on Kv1.5
mRNA expression. GH
cells were treated with 50 mM KCl for 3 h in the presence or absence of 0.5 µM nimodipine (NIM) or nifedipine (NIF). n = 5 in A, n = 4 in B. KCl
significantly decreased the level of Kv1.5 mRNA in the absence of
dihydropyridines (p < 0.01 in A and B),
in the presence of nimodipine (p < 0.05), and in the
presence of nifedipine (p <
0.01).
Figure 4:
Ca chelators do not
block the effect of depolarization. After 5-min preincubation with 2
mM BAPTA or 10 mM EGTA, 50 mM KCl was added
to the culture medium. RNA was prepared 3 h later (n =
5 for A, n
6 for B). KCl (K)
significantly lowered expression of Kv1.5 mRNA in controls (p < 0.01 in A and B), in the presence of BAPTA (p < 0.01), and in the presence of EGTA (p <
0.05). Because of the large difference in variance between the EGTA and
EGTA + K data, a one-tailed Wilcoxan signed rank test was used for
that comparison.
Treating
GH cells with elevated K
is known to
produce complex effects on cytoplasmic pH(33, 43) .
Alkalinization responses in GH
and related
GH
C
cells are blocked by amiloride or by
substituting extracellular Na
with organic
cations(33, 43) . We found that application of 200
µM amiloride had no significant effect on basal or
K
-induced regulation of Kv1.5 mRNA expression (Fig. 5). A more specific inhibitor, ethylisopropylamiloride,
also had no effect (data not shown). Likewise, substituting
extracellular NaCl with N-methylglucamine HCl did not prevent
K
-induced down-regulation of Kv1.5 mRNA (Fig. 5). Thus, neither Na
-H
exchange nor influx of Na
is required for the
effect of depolarization.
Figure 5:
Depolarization down-regulates Kv1.5 mRNA
in the presence of 200 µM amiloride or after replacement
of bath Na with N-methylglucamine. KCl
elevation for 3 h (K) reduced Kv1.5 mRNA levels in the
presence of amiloride (Amil.) (p < 0.01, n = 5) or after replacement of extracellular Na
with N-methylglucamine (NMG) (n = 3).
Figure 6:
Sustained application of
K is required for maximal down-regulation of Kv1.5
mRNA. GH
cells were initially treated with medium
supplemented with 50 mM KCl for 1 h. At 1 h, the medium was
replaced with either fresh standard tissue culture solution or with the
KCl-supplemented medium. Both groups were then harvested at 3 and 5 h (n = 3).
We then tested if induction of IEG products or other proteins are
required for down-regulating Kv1.5 mRNA. We found that K treatment did not significantly affect Fos protein expression in
GH
cells (data not shown). However, other IEG products
might participate in the depolarization response. Therefore, we blocked
the expression of all proteins with cycloheximide. Under our
conditions, cycloheximide inhibited
[
S]methionine incorporation into GH
cell protein by >95%. This inhibitor increased Kv1.5 mRNA as
expected(4) . However, cycloheximide treatment did not prevent
down-regulation by elevated K
(Fig. 7). Hence,
the regulation of Kv1.5 mRNA does not require protein synthesis.
Rather, sustained depolarization acts by a more direct mechanism to
decrease the channel mRNA level.
Figure 7:
Depolarization-induced down-regulation of
Kv1.5 mRNA does not require protein synthesis. GH cells
were treated for 3 h with 250 µM cycloheximide (CHX) alone or in the presence of 50 mM KCl (K). KCl significantly reduced channel message in the presence (p < 0.05) or absence of cycloheximide (p <
0.01) (n = 5).
Figure 8:
Depolarization inhibits Kv1.5 gene
transcription. A, autoradiogram showing nuclear run-on results
for -actin, Kv1.5, and a vector control after treatment with 50
mM KCl for different periods of time. B,
densitometric measurement of Kv1.5 gene transcription normalized to
actin gene transcription. Note that depolarization for 1 h
significantly reduced Kv1.5 gene transcription compared to control (p < 0.05, two-tailed Bonferroni test, n =
4).
Figure 9:
Depolarization of GH cells for
8 h decreases Kv1.5 protein expression. Concentrations shown indicate
the amount of KCl added to standard tissue culture medium. A,
immunoblot from one experiment with the 76-kDa band used for
quantitation by densitometry indicated. B, quantitation of
results from four experiments is shown. Single and double
asterisks indicate p < 0.05 and p
0.01,
respectively.
K channels influence the resting potential
and the shape and frequency of action potentials in excitable cells.
Electrical activity can in turn alter gene expression in neurons,
endocrine cells, and
muscle(34, 35, 36, 37) . Therefore,
we were interested in whether membrane potential might serve as a
feedback signal to control K
channel gene expression.
Thus, we examined KCl regulation of K
channel
expression in GH
clonal pituitary cells. Our results
establish that KCl acts via membrane depolarization to down-regulate
Kv1.5 mRNA in pituitary cells without affecting expression of Kv1.4 or
Kv2.1 mRNAs. Furthermore, we showed that the decrease in channel
message is due to inhibition of transcription and is independent of
induction of immediate early genes. Finally, we demonstrated for the
first time that depolarization rapidly alters K
channel protein expression. Our previous studies found that
changes in Kv1.5 protein levels correlate with alterations in delayed
rectifier K
current(4, 7) . Thus, we
suggest that depression of Kv1.5 gene expression by membrane
depolarization might produce a long-term enhancement of excitability
that could in turn promote secretion by pituitary cells.
Many
previous studies have indicated that effects of membrane depolarization
on expression of mRNAs encoding immediate early gene products,
nicotinic receptors, and neuropeptides are mediated by Ca influx through voltage-dependent Ca
channels(34, 35, 36, 37) .
Indeed, it was recently suggested that K
channel mRNA
levels in cultured cardiomyocytes and clonal pituitary cells is altered
by such a mechanism(5, 6) . However, we found that
chelating extracellular Ca
or blocking L-type
Ca
channels with dihydropyridines did not inhibit
down-regulation of Kv1.5 mRNA expression in GH
cells. Thus,
these studies rule out Ca
influx as the signal in
depolarization-induced down-regulation of Kv1.5 mRNA. Since
Ca
influx into the cytoplasm is required for hormone
secretion, these experiments also exclude autocrine effects caused by
depolarization-induced secretion. Our experiments with Na
substitution and amiloride analogs demonstrated that
Na
influx and Na
-H
exchange are also not involved in the action of membrane
depolarization. Thus, the effect of depolarization does not appear to
be mediated by well characterized effects of this stimulus. Rather,
depolarization of pituitary cells must act via a novel
Ca
-independent mechanism to specifically inhibit
transcription of the Kv1.5 gene without changing Kv1.4 and Kv2.1 mRNA
expression.
Reducing channel mRNA levels will only alter
excitability if it results in less channel protein expression.
Voltage-gated Na and Ca
channels
turn over slowly (t
1
day)(38, 39) . Hence, decreasing the concentrations of
mRNAs encoding those channels for 8 h would not be expected to markedly
affect the expression of these channel proteins. In contrast, we
recently demonstrated that Kv1.5 protein turns over with a half-life of
4 h in GH
cells. Thus, steroid hormone-induced
increases in Kv1.5 mRNA can act within hours to significantly enhance
channel protein expression(4) . Here we reported that
depolarization-induced down-regulation of Kv1.5 mRNA produces a
significant reduction in Kv1.5 protein within 8 h. Thus, Kv1.5 protein
expression can be rapidly up- or down-regulated in clonal pituitary
cells.
Depolarization might influence voltage-gated K channel gene expression under a variety of circumstances. While
some endocrine, neuronal, and muscle cells are typically electrically
silent, many spontaneously produce beating or bursting action potential
activity. Hence, long-term differences in basal action potential
activity might influence the resting level of K
channel gene expression via a cumulative effect of membrane
potential. Likewise, the large changes in electrical activity seen in
these cell types that occur during development, intense physiological
responses (e.g. secretion by the pituitary in response to
stress), with arrhythmias in the heart, and with epileptic seizures in
the brain, could also act via depolarization to alter channel
expression. Indeed, this mechanism may be responsible for
seizure-induced down-regulation of K
channel mRNAs in
brain(40) . Therefore, it is possible that intense electrical
activity induced by physiological or pathological conditions acts via
depolarization-induced changes in Kv1.5 K
channel gene
transcription and protein expression to produce long-term changes in
excitability. Interestingly, homomeric Kv1.5 channels are sensitive to
some antiarrhythmics and an
antihistamine(15, 16, 17, 18) .
Thus, the effect of electrical activity on K
channel
expression might also produce extended changes in the efficacy of these
therapeutic drugs.