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.

MATERIALS AND METHODS

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.

RESULTS

Specific Down-regulation of Kv1.5 K Channel mRNA by Membrane Depolarization

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.

Inhibition of Kv1.5 mRNA Expression Does Not Require Ca or Na Influx or Na-H Exchange

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 GHC 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).

Depolarization Must Be Sustained and Does Not Require Protein Synthesis to Affect Kv1.5 mRNA

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).

Depolarization Inhibits Kv1.5 Gene Transcription

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).

Kv1.5 Protein Expression Is Inhibited by Depolarization

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.

DISCUSSION

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.

FOOTNOTES

*

This research was supported by a postdoctoral fellowship from the American Heart Association (PA affiliate) (to K. T.), National Institutes of Health Grant NS 29876 and a grant from the Muscular Dystrophy Association (to J. S. T.), and by National Institutes of Health Grant NS 29804, a Klingenstein fellowship in the Neurosciences, and an American Heart Association (PA affiliate) grant-in-aid (to E. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence and reprint requests should be addressed: Dept. of Pharmacology/E1355 BST, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-648-9486; Fax: 412-648-1945; Levitan{at}bns.pitt.edu.

()

The abbreviations used are: IEG, immediate early gene; TEA, tetraethylammonium; 4-AP, 4-aminopyridine; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid.

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