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17β-Estradiol at Physiological Concentrations Augments Ca²⁺-Activated K⁺ Currents via Estrogen Receptor β in the Gonadotropin-Releasing Hormone Neuronal Cell Line GT1-7

Department of Physiology (I.N., H.I., Y.S., M.K.), Nippon Medical School, Tokyo 113-8602, Japan; and Department of Biophysics and Biochemistry (K.U.-T., K.S.), School of Science, University of Tokyo, Tokyo 113-0033, Japan

Address all correspondence and requests for reprints to: Dr. Masakatsu Kato, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo, Tokyo 113-8602, Japan. E-mail: mkato{at}nms.ac.jp.

	Abstract

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Estrogens play essential roles in the neuroendocrine control of reproduction. In the present study, we focused on the effects of 17β-estradiol (E2) on the K⁺ currents that regulate neuronal cell excitability and carried out perforated patch-clamp experiments with the GnRH-secreting neuronal cell line GT1-7. We revealed that a 3-d incubation with E2 at physiological concentrations (100 pM to 1 nM) augmented Ca²⁺-activated K⁺ [K(Ca)] currents without influencing Ca²⁺-insensitive voltage-gated K⁺ currents in GT1-7 cells. Acute application of E2 (1 nM) had no effect on the either type of K⁺ current. The augmentation was completely blocked by an estrogen receptor (ER) antagonist, ICI-182,780. An ERβ-selective agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile, augmented the K(Ca) currents, although an ER-selective agonist, 4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol, had no effect. Knockdown of ERβ by means of RNA interference blocked the effect of E2 on the K(Ca) currents. Furthermore, semiquantitative RT-PCR analysis revealed that the levels of BK channel subunit mRNAs for and β4 were significantly increased by incubating cells with 300 pM E2 for 3 d. In conclusion, E2 at physiological concentrations augments K(Ca) currents through ERβ in the GT1-7 GnRH neuronal cell line and increases the expression of the BK channel subunit mRNAs, and β4.

	Introduction

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THE GnRH NEUROSECRETORY system constitutes the final common pathway in the central regulation of reproduction. For the normal functioning of this system, the steroid hormone 17β-estradiol (E2) is indispensable. Despite the importance of E2 in this system, its mechanisms of action are largely unknown (1). E2 acts either through two subtypes of nuclear estrogen receptors (ERs), ER and ERβ, or directly on membrane proteins, including a hypothetical membrane ER (2). Modulation of K⁺ channels by E2 has been reported. E2 hyperpolarizes neurons of the medial amygdala (3) and preoptic area (4) by increasing their K⁺ permeability and depolarizes hypothalamic neurons by decreasing K⁺ permeability (5). E2 rapidly alters the firing patterns of primate GnRH neurons in culture (6). The acute application of E2 increases the inward currents and decreases the outward currents of hypothalamic neurons (7). E2 acutely modulates the function of large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channels either through a cGMP-dependent mechanism, as in coronary artery endothelial cells (8), or by binding to the auxiliary β1-subunit of BK channels, as in the oocyte expression system (9). These actions are considered to be nongenomic and appear only at nanomolar to micromolar concentrations of E2, which exceed the concentrations of rodent plasma E2 (10^–11 to 10^–10 M) (10, 11). In addition, implantation of E2 pellets in ovariectomized mice decreased both the delayed rectifier K⁺ and A currents in GnRH neurons (12), suggesting a long-term effect of E2.

An immortalized GnRH-secreting neuronal cell line, GT1, generated by genetically targeted tumorigenesis in transgenic mice, is thought to preserve many of the characteristics of native GnRH neurons (13). These neurons generate spontaneous action potentials, exhibit transient oscillations in intracellular Ca²⁺ concentration (14, 15), and secrete GnRH in a pulsatile manner (16, 17). Moreover, GT1-7 cells exhibit K⁺ currents, including delayed rectifier K⁺ currents, A currents, inward rectifier K⁺ currents, BK currents (18, 19), and small-conductance Ca²⁺-activated K⁺ (SK) currents (20) and express both ER and ERβ (21, 22). In addition, E2 directly represses GnRH gene expression in GT1-7 cells (22). Thus, this cell line is suitable for studying the action of estrogens on K⁺ channels. In the present study, we analyzed the action of E2 on the function of K⁺ channels and found that Ca²⁺-activated K⁺ [K(Ca)] currents are positively modulated by physiological concentrations of E2 via ERβ. No acute effects of E2 on K(Ca) currents were observed at physiological concentrations.

	Materials and Methods

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Cell culture
GT1-7 cells (provided by Dr. R. Weiner, University of California, San Francisco) were cultured in DMEM (without phenol red; Irvine Scientific, Santa Ana, CA) supplemented with 1 mM sodium pyruvate, 24 mM NaHCO₃, 4 mM L-glutamine, 10% fetal bovine serum (JRH Biosciences, Lenexa, KS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA). The fetal bovine serum contained 72 pM E2; therefore, the concentration of E2 in the culture media without E2 supplementation was 7.2 pM. The cultures were maintained at 37 C in a water-saturated atmosphere of 95% air and 5% CO₂. The culture medium was changed every 3–4 d, and the cells were passaged every 1–2 wk and used in experiments within 10 passages. For electrophysiological experiments, the cells were plated onto poly-L-lysine-coated coverslips and cultured for 3 d with or without selective ER modulators.

Treatment of cells with E2 and ER modulators
Cells were incubated with E2 (10 pM to 10 nM) for 3 d. The effect of the ER antagonist ICI-182,780 (ICI) was determined by incubating cells in medium containing ICI (0.3–10 µM) and E2 (300 pM) for 3 d. 4,4',4''-[4-Propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol (PPT) was used as an ER-selective agonist and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was used as an ERβ-selective agonist. Cells were incubated with PPT (1–10 nM) or DPN (0.1–10 nM) for 3 d to determine the effects of these agonists.

RNA interference
Using Lipofectamine 2000 (Invitrogen), GT1-7 cells were cotransfected with pCAGIPuro-EGFP (0.3 µg/ml), encoding enhanced green fluorescent protein and puromycin-resistance genes, and 50 nM small interfering RNA (siRNA) against either mouse ER or ERβ, according to the manufacturer’s protocol. The sequences of the siRNAs were designed according to the guidelines reported by Ui-Tei et al. (23): 5'-CUGGUUCAUAUGAUCAACUGG-3' for sense ER siRNA (1146S); 3'-AGUUGAUCAUAUGAACCAGCU-5' for antisense ER siRNA (1146A); 5'-GGAACUGGUGCACAUGAUUGG-3' for sense ERβ siRNA (775S); and 3'-AAUCAUGUGCACCAGUUCCUU-5' for antisense ERβ siRNA (775A). After siRNA transfection, we prepared the cells for each set of experiments according to the following procedures. For electrophysiological experiments, cells were incubated with E2 for 3 d after overnight preincubation without E2. For RT-PCR, the cells were incubated with puromycin (1 µg/ml) for 2 d to isolate transfected cells after overnight preincubation without E2 and puromycin.

RT-PCR
Total RNA was extracted from GT1-7 cells using RNeasy Mini kits (QIAGEN, Valencia, CA) following the manufacturer’s instructions. Total RNA was then treated with 5 U RNase-free DNase I (Ambion, Austin, TX). The concentration of total RNA was quantified by measurement of the absorption at 260 nm. Total RNA was reverse-transcribed into first-strand cDNA using an oligo-dT primer. Reaction mixtures (final volume, 25 µl) contained 5 µg total RNA, 1x RT buffer, 1 mM dNTP mixture, 1 µg oligo-(dT)₁₅ primer (Promega, Madison, WI), 20 U RNasin Plus, (Promega), and 100 U Maloney murine leukemia virus reverse transcriptase (ReverTra Ace; Toyobo Bio, Osaka, Japan). The reaction was carried out at 42 C for 1 h and stopped by heating at 72 C for 15 min. cDNA was treated with 4 U RNase H (Takara Bio, Shiga, Japan) at 37 C for 30 min and stored at –20 C.

PCR was performed in a 20-µl reaction mixture comprising cDNA corresponding to 50 ng total RNA, 1x PCR buffer, 0.2 mM dNTP mixture, 0.2 µM forward and reverse primers, and 0.63 U Blend Taq plus (Toyobo Bio). The PCR conditions were 94 C for 2 min, followed by 18–38 cycles of 94 C for 30 sec, a suitable temperature for each primer pair for 20 sec, 72 C for 30 sec, and finally, 72 C for 5 min. The sequences of oligonucleotide primers used in this study are listed in Table 1. To avoid amplifying any contaminating genomic DNA, the primer pairs were designed from different exons. The optimal number of PCR cycles was determined over a range of cycles to identify an exponentially linear range of amplification for each transcript extracted from GT1-7 cells. PCR products (5 µl) were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining under UV irradiation. Gel images were captured using a FAS-III system (Toyobo Bio).

DNA sequencing
PCR products were extracted from agarose gels using a Wizard SV Gel and PCR Clean-up system (Promega), and cloned into pGEM-T-Easy vectors (Promega). Sequencing reactions were performed using a BigDye Terminator version 3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA). Fluorescent signals were detected using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Electrophysiology
A List EPC-9 patch-clamp system (HEKA Elektronik, Lambrecht, Germany) was used for recordings and data analyses. Whole-cell currents were recorded using a perforated patch-clamp configuration with 50 µg/ml amphotericin B (Seikagaku Corp, Tokyo, Japan) (24, 25) at room temperature (25 C). The patch electrodes were made of borosilicate glass capillaries and had a resistance of 5–10 M. The indifferent electrode consisted of an Ag-AgCl wire connected to the bath solution via an agar bridge. For the recording of K⁺ currents, the pipette solution consisted of 95 mM K-aspartate, 47.5 mM KCl, 1 mM MgCl₂, 0.1 mM EGTA, 10 mM HEPES, and 2 mM ATP-Mg (pH 7.2; osmolality, 270 mOsm). The extracellular solution consisted of 137.5 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 0.8 mM MgCl₂, 10 mM glucose, 20 mM HEPES, and 0.6 mM NaHCO₃ (pH 7.4; osmolality, 300 mOsm). Na⁺ currents were blocked by 0.3 µM tetrodotoxin (Sankyo Co., Ltd., Tokyo, Japan). The difference in the osmolality between the pipette solution and extracellular solution somehow helped to form a stable giga-seal in the perforated patch configuration. For the recording of Ca²⁺ currents, K⁺ in the pipette solution was replaced with Cs⁺. The extracellular solution consisted of 106.5 mM NaCl, 5 mM CsCl, 0.8 mM MgCl₂, 10 mM CaCl₂, 10 mM glucose, 20 mM HEPES, 0.6 mM NaHCO₃, and 10 mM tetraethylammonium chloride (pH 7.4; osmolality, 300 mOsm). Na⁺ currents were blocked by 0.3 µM tetrodotoxin.

The currents were filtered at 2.3 kHz, digitized at 10 kHz, and recorded. Data were collected only when the series resistance was stable and less than 25 M. Up to 70% of the series resistance was electronically compensated. Capacitive and leak currents were subtracted by means of the P/4 protocol. Cell capacitance was 9.0 ± 0.2 pF (mean ± SEM, n = 166). The liquid junction potential was not compensated. K⁺ currents were activated by 50-msec voltage steps from –60 to +60 mV in 10-mV increments from a holding potential of –90 mV at 0.2 Hz (Fig. 1A). The K⁺ current values were determined as the means for the last 2.5 msec of the voltage pulse. After-hyperpolarization currents (I_AHP) were activated by a voltage protocol comprising a 100-msec voltage pulse to +30 mV from a holding potential of –90 mV, followed by a 5-sec voltage step to –50 mV. The voltage pulse was applied every 30 sec (Fig. 4C). Ca²⁺ currents were activated by 100-msec voltage steps from –60 to +60 mV in 10-mV increments from a holding potential of –100 mV at 0.2 Hz (Fig. 5A). The peak Ca²⁺ currents were determined for the analysis.

View larger version (28K): [in this window] [in a new window]   FIG. 1. K(Ca) currents and Ca2+-insensitive K+ currents in GT1-7 cells. The K+ currents were activated by 50-msec voltage pulses (–60 to +60 mV in 10-mV increments) from a holding potential of –90 mV. A, The voltage protocol and current traces activated by voltage pulse to +60 mV are shown. Under the control conditions, Ni2+ (100 µM) and Cd2+ (200 µM) slightly reduced the K+ currents. However, Ni2+ and Cd2+ clearly reduced the currents in cells incubated with 300 pM E2 for 3 d. B, The effects of Ni2+ and Cd2+ are collectively shown in the current-voltage relationship of the K+ currents in the control cells (n = 10) and in the cells incubated with 300 pM E2 for 3 d (n = 10). C, The effects of Ni2+ and Cd2+ and washout are shown for individual cells. Three cells were lost during the washout. Therefore, only seven data points for washout are shown. *, P < 0.05 vs. normal medium; **, P < 0.01 vs. normal medium using the Wilcoxon signed-rank test.

View larger version (19K): [in this window] [in a new window]   FIG. 4. The K(Ca) channel blocker ChTX reduced the K+ currents. A, Current-voltage relationships of the K+ currents in control (n = 7) and 300 pM E2-treated cells (n = 7) are shown. K+ currents were evoked by voltage steps from –60 to +60 mV in 10-mV increments from a holding potential of –90 mV. Data for Ni2+ and Cd2+ are not shown for clarity. B, ChTX (100 nM)-sensitive currents (black bars), Ni2+ (100 µM)- and Cd2+ (200 µM)-sensitive currents [white bars, K(Ca) currents] and apamin-sensitive currents (gray bar) are shown. Approximately 80% of the K(Ca) currents were ChTX sensitive in control cells (n = 7) and 70% in cells incubated with 300 pM E2 for 3 d (n = 7). Apamin-sensitive currents comprised 7.6 ± 6.5% of the K(Ca) currents in cells incubated with 300 pM E2 for 3 d (n = 5). C, Voltage-protocol and example traces for IAHP. The currents were activated by a 100-msec voltage pulse to +30 mV from a holding potential of –90 mV, followed by a 5-sec voltage step to –50 mV. IAHP were hardly observed in cells incubated with or without E2 for 3 d. *, P < 0.05 vs. normal medium; **, P < 0.01 vs. normal medium using the Wilcoxon signed-rank test.

View larger version (32K): [in this window] [in a new window]   FIG. 5. Voltage-gated Ca2+ currents. Ca2+ currents were activated by 100-msec voltage pulses (–60 to +60 mV in 10-mV increments) from a holding potential of –100 mV. Example current traces activated by +20-mV voltage pulses are shown. B, Current-voltage relationship of the peak Ca2+ currents. There were no significant differences between the currents recorded from control cells (n = 10) and those recorded from E2-treated cells (300 pM; n = 10). Ni2+ and Cd2+ blocked the Ca2+ currents in the both cell groups ().

Statistical analysis
Data are presented as the means ± SEM. The differences were analyzed by a one-way ANOVA followed by the Tukey-Kramer multiple comparison test, Wilcoxon signed-rank test, or t test. P < 0.05 was considered statistically significant.

	Results

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K(Ca) currents and Ca²⁺ currents
The activation of the K⁺ currents started at –10 mV, and the amplitudes increased with depolarization (Fig. 1B). The K⁺ currents were slightly reduced by the addition of Ni²⁺ (NiCl₂; 100 µM) and Cd²⁺ (CdCl₂; 200 µM), which completely block Ca²⁺ channels. Ni²⁺ and Cd²⁺ clearly reduced the amplitude of the K⁺ currents in cells incubated with 300 pM E2 for 3 d. The effects of Ni²⁺ and Cd²⁺ were almost completely abolished on washout (Fig. 1C). The rates of recovery from the maximal amplitude were 97.2 ± 1.4% in control cells and 97.4 ± 0.8% in cells incubated with 300 pM E2. These Ni²⁺- and Cd²⁺-blockable K⁺ currents are referred to as the K(Ca) currents, and the remaining currents are referred to as the Ca²⁺-insensitive K⁺ currents. The K(Ca) currents were not augmented by a 1-d incubation with E2 (data not shown). Acute application (5 min) of 1 nM E2 had no effect on the total K⁺ currents in the cell groups incubated with or without 300 pM E2 for 3 d (data not shown).

To determine the concentration-response relationship of E2, incubations were carried out with 10 pM to 10 nM E2 for 3 d. E2 had no effect on the Ca²⁺-insensitive K⁺ currents (Fig. 2), whereas it augmented the K(Ca) currents in the concentration range of 100 pM to 1 nM (Fig. 3). The EC₅₀ was 56.5 pM. The effects of E2 were observed at +30 mV to +60 mV but not at potentials more negative than +30 mV (Fig. 3A). In control cells, the K(Ca) currents were 4.8 ± 0.5 pA/pF at +20 mV and 3.3 ± 0.8 pA/pF at +60 mV. It is to be noted that the currents from the control cells were not increased at potentials more positive than +20 mV. In cells incubated with 300 pM E2, the K(Ca) currents were 7.7 ± 1.5 pA/pF at +20 mV and 12.3 ± 1.2 pA/pF at +60 mV. A BK channel blocker, ChTX (100 nM), slightly reduced the K⁺ currents in control cells and clearly reduced those in cells incubated with 300 pM E2 (Fig. 4, A and B). This inhibitory effect by ChTX was not observed when Ni²⁺ and Cd²⁺ were applied before ChTX (data not shown). The ChTX-sensitive component comprised 80.1 ± 7.8% of the K(Ca) currents in control cells and 70.1 ± 5.7% of the K(Ca) currents in cells incubated with E2 (Fig. 4B). On the other hand, the SK channel blocker apamin (100 nM) did not reduce the K⁺ currents at +60 mV in cells incubated with 300 pM E2 (Fig. 4B). Furthermore, I_AHP were hardly observed in cells incubated with or without 300 pM E2 (Fig. 4C).

View larger version (20K): [in this window] [in a new window]   FIG. 2. E2 had no effect on the Ca2+-insensitive K+ currents, which are the remaining currents after application of Ni2+ and Cd2+. A, The current-voltage relationships of the Ca2+-insensitive K+ currents are shown for GT1-7 cells incubated with 300 pM E2 (, n = 10) and without 300 pM E2 (, n = 10). The data are repeated from Fig. 1B. B, Concentration-response relationship of E2 on the Ca2+-insensitive K+ currents at +60 mV (n = 7–10).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Effect of E2 on the K(Ca) currents. The K(Ca) currents were obtained by subtracting the Ni2+- and Cd2+-insensitive currents from the total K+ currents. Cells were incubated with E2 for 3 d. A, Current-voltage relationships of the K(Ca) currents in the control cells (, n = 10) and in the cells incubated with 300 pM E2 for 3 d (, n = 10). E2 (300 pM) augmented K(Ca) currents at +30 to +60 mV. B, Concentration-response relationship of E2 on K(Ca) currents (n = 7–10). E2 had no effect on the K(Ca) currents activated at +20 mV. It is to be noted that the maximal amplitude of voltage-gated Ca2+ currents is attained around this voltage. The K(Ca) currents activated at +60 mV were clearly augmented by physiological concentrations of E2. However, 10 nM E2 had no statistically significant effect on the K(Ca) currents. There was no statistically significant difference between the current in cells incubated with 1 nM E2 and that with 10 nM E2. **, P < 0.01 vs. control (0 pM E2) using the Tukey-Kramer multiple comparison test.

Effects of ER agonists and antagonist
To determine whether E2 acts through ERs, an ER antagonist, ICI, was added to the culture medium with 300 pM E2. ICI blocked the effect of E2 on K(Ca) currents in a concentration-dependent manner, with an IC₅₀ of 1.2 µM (Fig. 6). To examine whether E2 affected either ER or ERβ, a selective agonist for each subtype of ER was used. The selective agonist for ERβ (DPN) augmented the K(Ca) currents, whereas that for ER (PPT) had no effect on K(Ca) currents (Fig. 7A). DPN significantly augmented the K(Ca) currents at +30, +50, and +60 mV, compared with control cells (Fig. 7A). The augmentation by DPN was observed at concentrations of 1–10 nM, but PPT in the same concentration range had no effect on K(Ca) currents (Fig. 7, A and B).

View larger version (15K): [in this window] [in a new window]   FIG. 6. The ER antagonist ICI blocked the E2-induced augmentation of the K(Ca) currents. A, Current-voltage relationships of the K(Ca) currents. The augmentation by 300 pM E2 (, n = 10) was completely blocked by 3 µM ICI (, n = 10). The data with 300 pM E2 alone are repeated from Fig. 3A. B, The concentration-response relationship of ICI in the presence of 300 pM E2 is shown at a voltage-pulse of +60 mV (n = 7–10). *, P < 0.05 vs. 300 pM E2 without ICI; **, P < 0.01 vs. 300 pM E2 without ICI using the Tukey-Kramer multiple comparison test.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Effects of selective ER agonists on the K(Ca) currents. A, The current-voltage relationships of the BK currents are collectively shown. The ER-selective agonist PPT had no effect on K(Ca) currents (, n = 10), whereas the ERβ-selective agonist DPN increased the BK currents at +30 mV, +50 mV and +60 mV (, n = 10). The control data (, n = 10) are repeated from Fig. 3A. B, Concentration-response relationships of PPT and DPN at +60 mV (n = 7–10). *, P < 0.05 vs. control; **, P < 0.01 vs. control using the Tukey-Kramer multiple comparison test.

Knockdown of ER and ERβ

View larger version (33K): [in this window] [in a new window]   FIG. 8. Effect of ER knockdown on K(Ca) currents. Both ER and ERβ were knocked down by siRNA. Control cells were transfected only with pCAGIPuro-EGFP, encoding enhanced green fluorescent protein. All transfected cells were cultured with 300 pM E2. A, The levels of mRNAs for the ERs were measured using RT-PCR in control (Cont.) and siRNA-treated cell groups targeted at ER (ER-KD) and ERβ (ERβ-KD). B, Current-voltage relationships of the K(Ca) currents. The facilitatory effect of E2 was not observed in ERβ-KD cells (, n = 7). However, augmentation by E2 of the K(Ca) currents was observed in both ER-KD (, n = 7) and control cells (, n = 7). *, P < 0.05 vs. control; **, P < 0.01 vs. control using the Tukey-Kramer multiple comparison test.

View larger version (33K): [in this window] [in a new window]   FIG. 9. RT-PCR analysis of the expression of BK channel - and β1- to β4-subunit mRNAs in GT1-7 cells. Mouse hypothalamus and testis were used as positive controls. PCRs were performed with (+) or without (–) cDNA. B, The levels of mRNAs for - and β4-subunits in GT1-7 cells were increased by treatment with 300 pM E2 for 3 d (black bars, n = 7) compared with control cells (white bars, n = 7). However, E2 had no effect on the level of mRNAs for β1- and β2-subunits. The expression levels of each BK channel mRNA relative to the level of GAPDH mRNA in the same sample were normalized to control values. *, P < 0.05 vs. control; **, P < 0.01 vs. control using the t test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is essential for the activation of K(Ca) channels. In the present experimental conditions, an increase in [Ca²⁺]_i is achieved by Ca²⁺ influx through voltage-gated Ca²⁺ channels. However, the Ca²⁺ channels are not the target of E2 action, because incubation with E2 did not augment the Ca²⁺ currents (Fig. 5).

Three types of K(Ca) currents are known (28), namely the SK currents, the intermediate-conductance K(Ca) (IK) currents, and the BK currents. Which type(s) of currents comprise the K(Ca) currents observed in the present experiments? Involvement of SK currents is unlikely, because the SK channel blocker apamin did not attenuate the K(Ca) currents, and the expression of either fast or slow after-hyperpolarization (AHP) current was very small (Fig. 4, B and C). Either current is carried through K(Ca) channels (29). Fast AHP currents in hippocampal and neocortical pyramidal neurons are carried through SK channels (30), and slow AHP currents in GnRH neurons are carried through SK channels (31, 32). In GT1-7 cells, lack of effect of apamin on whole-cell currents induced by depolarizing voltage pulses has been reported (19), whereas apamin is reported to block the outward currents induced by mobilization of intracellular Ca²⁺, indicating the presence of SK channels (20). The discrepancy in the effect of apamin might be due to difference in the experimental protocol. The IK currents, like the SK currents, are voltage independent and solely activated by increases in Ca²⁺ concentration. The K(Ca) currents in the control were not increased at potentials more positive than +20 mV (Fig. 3A), indicating the possible involvement of IK currents. Activation of the IK currents must be closely related to an influx of Ca²⁺ through voltage-gated Ca²⁺ channels in the present experimental conditions. The Ca²⁺-influx reached a maximum at +10 to +20 mV and declined at more positive potentials due to a decrease in the driving force (Fig. 5). In addition, IK currents are also blocked by the BK channel blocker ChTX. Expression of IK channels in brain tumor tissues has been reported, but there are no reports of IK channel expression in normal brain tissues (33). Because GT1-7 cells are generated by genetically targeted tumorigenesis (13), these cells possibly express IK channels.

K(Ca) currents recorded from E2-treated cells increased voltage dependently (Fig. 3) and were blocked by ChTX (Fig. 4, A and B). These findings indicate that the majority of currents at potentials more positive than +20 mV are carried through BK channels, because the BK channels are dually activated by membrane depolarization and increases in [Ca²⁺]_i (34, 35). At potentials between +20 mV and +60 mV, depolarization of the membrane potential decreases the influx of Ca²⁺. These two interrelated factors probably cause a small increase in the activation of BK channels at these potentials. The conductance values, calculated by using the value of –90 mV as the equilibrium potential of K⁺, were 0.63 pS at +20 mV and 0.74 pS at +60 mV in cells incubated with E2 and 0.39 pS at +20 mV and 0.2 pS at +60 mV in control cells. Taken together, a plausible explanation is that GT1-7 cells weakly express BK channels together with IK channels in the control condition, and incubation with E2 increases the functional expression of BK channels. Alternatively, the function of BK channels might be inhibited through an unknown mechanism, and E2 might remove this inhibition. In any case, the precise mechanism remains to be elucidated.

BK channels are composed of pore-forming -subunits and auxiliary β-subunits (36, 37). To date, one type of -subunit and four types of β-subunit (β1–β4) have been identified, different combinations of which form functional BK channels with different characteristics (38, 39, 40, 41, 42). GT1-7 cells expressed -, β1-, β2-, and β4-subunits, similar to the expression pattern seen in the mouse hypothalamus (Fig. 9A) and rat GnRH neurons (our unpublished data). The β3-subunit, which is expressed mainly in testis, pancreas, and spleen (42), was not detected in GT1-7 cells. Blockade by ChTX was incomplete (80%) in the present experiments (Fig. 4B). This may be due to the expression of the β4-subunit in GT1-7 cells, because coexpression of the β4-subunit with the -subunit in Xenopus oocytes reduces sensitivity to blockade by ChTX (40). Interestingly, the mRNA and proteins for the β1-subunit are increased in myometrial tissues after prolonged exposure of ovariectomized mice to E2 (43, 44), suggesting that E2 modulates BK channels at the level of transcription. The present results also demonstrated that E2 increased the level of mRNA for the - and β4-subunits, without affecting those for the β1- and β2-subunits (Fig. 9B). An increase in the expression level of the -subunit is likely to result in an increase in the number of BK channels. Increased expression of the β4-subunit probably attenuates the sensitivity of the channel to ChTX. These transcriptional regulations of BK channels by E2 are likely to be at least in part the cause of an increase in K(Ca) currents by E2, although the precise mechanism remains to be elucidated. The concentration-response relationship of E2 showed a bell shape with a maximal effect at 300 pM and no significant effect at 10 nM (Fig. 3B), as known for the effect of E2 on LH secretion by rat pituitary cells (45) and uptake of dopamine into fetal rat hypothalamic cells (46).

In contrast to previous reports that demonstrated acute effects of E2 on K⁺ currents (3, 4, 5), including BK currents (8, 9), we observed only a long-term effect (3 d) of E2. This difference is probably due to the concentrations of E2. We applied E2 at sub-nanomolar concentrations (Fig. 3), whereas the previous authors used E2 at nanomolar to micromolar concentrations. In rats and mice, plasma E2 levels range between a minimum of 10^–11 M during estrus and a maximum of about 10^–10 M during estrus (10, 11). Thus, under physiological conditions, cells are exposed to estrogens at sub-nanomolar concentrations. We did not, therefore, examine the effects of E2 at concentrations higher than this range, although locally synthesized E2 could exert acute actions at higher concentrations.

In conclusion, the present results indicate that E2 at physiological concentrations augments K(Ca) currents via ERβ, at least partly by increasing the transcription of BK channel genes, thereby modifying the excitability of GT1-7 cells. A large part of the K(Ca) currents is likely to comprise BK currents, although the possible involvement of IK currents cannot be ruled out. Finally, the present results suggest the possibility that expression of BK channels in GnRH neurons is directly regulated by E2 via ERβ, because GnRH neurons express ERβ (47, 48, 49).

	Acknowledgments

 
We are grateful to Drs. Yoshie Hiraizumi, Nobuyuki Tanaka, and Chengzhu Yin and Ms. Sumiko Usui for their technical support.

	Footnotes

 
This research was supported in part by JSPS Grants-in-Aid for Scientific Research (16590180, 18390070 and 18590226), a MEXT Grant-in-Aid for Scientific Research on Priority Areas (1686210), and an Ishidsu Shun Memorial Scholarship.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 25, 2007

Abbreviations: AHP, After-hyperpolarization; BK, large-conductance Ca²⁺- and voltage-activated K⁺; ChTX, charybdotoxin; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; E2, 17β-estradiol; ER, estrogen receptor; ERβ-KD, ERβ knockdown; I_AHP, after-hyperporalization current; ICI, ICI-182,780; IK, intermediate-conductance Ca²⁺-activated K⁺; K(Ca), Ca²⁺-activated K⁺; PPT, 4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol; siRNA, small interfering RNA; SK, small-conductance Ca²⁺-activated K⁺.

Received June 8, 2007.

Accepted for publication October 16, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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