17²-Estradiol at Physiological Concentrations Augments Ca2+-Activated K+
Currents via Estrogen Receptor ² in the Gonadotropin-Releasing Hormone
Neuronal Cell Line GT1-7
Ichiro Nishimura, Kumiko Ui-Tei, Kaoru Saigo, Hirotaka Ishii, Yasuo Sakuma and Masakatsu Kato
Endocrinology 2008 149:774-782 originally published online Oct 25, 2007; , doi: 10.1210/en.2007-0759

To subscribe to Endocrinology or any of the other journals published by The Endocrine
Society please go to: http://endo.endojournals.org//subscriptions/

Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online

0013-7227/08/$15.00/0
Printed in U.S.A.

Endocrinology 149(2):774 –782
Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2007-0759

17␤-Estradiol at Physiological Concentrations Augments
Ca2ⴙ-Activated Kⴙ Currents via Estrogen Receptor ␤ in
the Gonadotropin-Releasing Hormone Neuronal Cell
Line GT1-7
Ichiro Nishimura, Kumiko Ui-Tei, Kaoru Saigo, Hirotaka Ishii, Yasuo Sakuma, and Masakatsu Kato
Department of Physiology (I.N., H.I., Y.S., M.K.), Nippon Medical School, Tokyo 113-8602, Japan; and Department ofBiophysics and Biochemistry (K.U.-T., K.S.), School of Science, University of Tokyo, Tokyo 113-0033, Japan
tive agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile, augmented the K(Ca) currents, although an ER␣-selective
agonist, 4,4ⴕ,4ⴖ-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol,
had no effect. Knockdown of ER␤ by means of RNA interference blocked the effect of E2 on the K(Ca) currents. Furthermore, semiquantitative RT-PCR analysis revealed that thelevels of BK channel subunit mRNAs for ␣ and ␤4 were significantly increased by incubating cells with 300 pM E2 for 3 d.
In conclusion, E2 at physiological concentrations augments
K(Ca) currents through ER␤ in the GT1-7 GnRH neuronal cellline and increases the expression of the BK channel subunit
mRNAs, ␣ and ␤4. (Endocrinology 149: 774 –782, 2008)

Estrogens play essential roles in the neuroendocrine controlof reproduction. In the present study, we focused on the effects of 17␤-estradiol (E2) on the Kⴙ currents that regulate
neuronal cell excitability and carried out perforated patchclamp experiments with the GnRH-secreting neuronal cellline GT1-7. We revealed that a 3-d incubation with E2 at physiological concentrations (100 pM to 1 nM) augmented Ca2ⴙactivated Kⴙ [K(Ca)] currents without influencing Ca2ⴙ-insensitive voltage-gated Kⴙ currents in GT1-7 cells. Acuteapplication of E2 (1 nM) had no effect on the either type of Kⴙ
current. The augmentation was completely blocked by an estrogen receptor (ER) antagonist, ICI-182,780. An ER␤-selec-Toocyte expression system (9). These actions are considered to
be nongenomic and appear only at nanomolar to micromolar
concentrations of E2, which exceed the concentrations ofrodent 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 ina 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 smallconductance Ca2⫹-activated K⫹ (SK) currents (20) and express both ER␣ and ER␤ (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 actionof E2 on the function of K⫹ channels and found that Ca2⫹activated K⫹ [K(Ca)] currents are positively modulated byphysiological concentrations of E2 via ER␤. No acute effectsof E2 on K(Ca) currents were observed at physiologicalconcentrations.

HE GnRH NEUROSECRETORY system constitutes the
final common pathway in the central regulation of
reproduction. For the normal functioning of this system, thesteroid hormone 17␤-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 subtypesof nuclear estrogen receptors (ERs), ER␣ and ER␤, 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) andpreoptic area (4) by increasing their K⫹ permeability anddepolarizes 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 bybinding to the auxiliary ␤1-subunit of BK channels, as in theFirst Published Online October 25, 2007
Abbreviations: AHP, After-hyperpolarization; BK, large-conductanceCa2⫹- and voltage-activated K⫹; ChTX, charybdotoxin; DPN, 2,3-bis(4hydroxyphenyl)-propionitrile; E2, 17␤-estradiol; ER, estrogen receptor;
ER␤-KD, ER␤ knockdown; IAHP, 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]trisphenol; siRNA, small interfering RNA; SK, small-conductance Ca2⫹activated K⫹.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving theendocrine community.

Materials and Methods
Cell culture
GT1-7 cells (provided by Dr. R. Weiner, University of California, San
Francisco) were cultured in DMEM (without phenol red; Irvine Scien-774

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

tific, 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 awater-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 electrophysiologicalexperiments, 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 ␮m) and E2 (300 pm) for 3 d. 4,4⬘,4⬙[4-Propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol (PPT) was used as anER␣-selective agonist and 2,3-bis(4-hydroxyphenyl)-propionitrile(DPN) was used as an ER␤-selective agonist. Cells were incubated with
PPT (1–10 nm) or DPN (0.1–10 nm) for 3 d to determine the effects of theseagonists.

RNA interference
Using Lipofectamine 2000 (Invitrogen), GT1-7 cells were cotransfected with pCAGIPuro-EGFP (0.3 ␮g/ml), encoding enhanced green
fluorescent protein and puromycin-resistance genes, and 50 nm smallinterfering RNA (siRNA) against either mouse ER␣ or ER␤, 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 ER␤ siRNA(775S); and 3⬘-AAUCAUGUGCACCAGUUCCUU-5⬘ for antisense ER␤
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 ␮g/ml) for 2 d to isolate transfected cells after
overnight preincubation without E2 and puromycin.

Endocrinology, February 2008, 149(2):774 –782

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 firststrand cDNA using an oligo-dT primer. Reaction mixtures (final volume,
25 ␮l) contained 5 ␮g total RNA, 1⫻ RT buffer, 1 mm dNTP mixture, 1
␮g 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-␮l reaction mixture comprising cDNA
corresponding to 50 ng total RNA, 1⫻ PCR buffer, 0.2 mm dNTP mixture, 0.2 ␮m 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 sequencesof 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 ␮l) were separated by electrophoresis on 2% agarosegels and visualized by ethidium bromide staining under UV irradiation.
Gel images were captured using a FAS-III system (Toyobo Bio).

Semiquantitative RT-PCR
The expression levels of BK channel subunit mRNAs in GT1-7 cells
incubated with or without E2 were measured by semiquantitative RTPCR. Total RNA was extracted from GT1-7 cells incubated for 3 d
without E2 for the control group and with 300 pm E2 for the experimentalgroup. RT-PCR was performed using the same method as describedabove. The number of PCR cycles was selected to be within the rangeof the linear amplification for each transcript: 16 cycles for GAPDH, 26
cycles for Kcnma1 (␣), 25 cycles for Kcnmb1 (␤1), 23 cycles for Kcnmb2(␤2), and 23 cycles for Kcnmb4 (␤4). A quantitative densitometry anal-

TABLE 1. Sequences of the primers employed for RT-PCR amplifications, amplicon length, annealing temperatures, and PCR cycles
Gene nameER␣
Forward
ReverseER␤
Forward
ReverseGAPDHForward
ReverseKcnma1
Forward
Reverse
Kcnmb1
Forward
Reverse
Kcnmb2
Forward
Reverse
Kcnmb3Forward
Reverse
Kcnmb4
Forward
ReversePrimer sequence (5⬘–3⬘)

Ampliconlength(bp)

Annealing
temperature(C)

PCR
cycles

404

59

28

586

65

38

359

62

18

537

58

30

331

61

28

625

66

26

222

67

35

589

63

26

TGGCTGGAGATTCTGATGATTGGT
ATGTGCCGGATATGGGAAAGAA
CTTTGACATGCTCCTGGCGACGACGGGAAGCGCAACGTGGGTAAGGTGAAGGTCGGTGTGAACGGATTTG
GGCGGAGATGATGACCCTTTTG
CGCCAGCCGTCCATCACACAGCCGGTAAATTCCAAAACAAAG
CAGTGGCCATGGGGAAGAAGCCTGGGGATATAGGAGCACTGTTGGTTT
CCAGTGGCCGGACCTCTTCATCTTACAGATTGCCACACCCCCAGCCATCATACGATCCCCATGCCTGCAGGTGTTCGTAAACCAAGGGGCCATCGGGACTGTAGACTCCGGCATCCTGTCGCTCTTCA
GGTCTCGGCTTCCCACAATCCTC

775

776

Endocrinology, February 2008, 149(2):774 –782

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

ysis was performed using CS analyzer software (Atto Corp., Tokyo,
Japan). The levels of mRNAs for BK channel subunits in each sample
were expressed as a ratio to the level of GAPDH in the same sample.
Values were normalized to control levels.

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 with50 ␮g/ml amphotericin B (Seikagaku Corp, Tokyo, Japan) (24, 25) atroom 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, 270mOsm). 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 ␮m 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 thepipette solution was replaced with Cs⫹. The extracellular solution consisted of 106.5 mm NaCl, 5 mm CsCl, 0.8 mm MgCl2, 10 mm CaCl2, 10mm 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 ␮m 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 electronicallycompensated. 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). TheK⫹ current values were determined as the means for the last 2.5 msecof the voltage pulse. After-hyperpolarization currents (IAHP) were activated by a voltage protocol comprising a 100-msec voltage pulse to ⫹30mV from a holding potential of ⫺90 mV, followed by a 5-sec voltage stepto ⫺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.

Chemicals
SNX-482, charybdotoxin (ChTX), and apamin were purchased fromthe Peptide Institute (Osaka, Japan). Nifedipine was purchased from
Sigma. ICI, PPT, and DPN were purchased from Tocris (Ellisville, MO).
All drugs were prepared immediately before use.

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 wasconsidered statistically significant.

Results
K(Ca) currents and Ca2⫹ currents

The activation of the K⫹ currents started at ⫺10 mV, andthe amplitudes increased with depolarization (Fig. 1B). The

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 ␮M) andCd2⫹ (200 ␮M) slightly reduced the K⫹ currents. However, Ni2⫹ andCd2⫹ 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 thecurrent-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 sevendata points for washout are shown. *, P ⬍ 0.05 vs. normal medium;**, P ⬍ 0.01 vs. normal medium using the Wilcoxon signed-rank test.

K⫹ currents were slightly reduced by the addition of Ni2⫹
(NiCl2; 100 ␮m) and Cd2⫹ (CdCl2; 200 ␮m), which completely
block Ca2⫹ channels. Ni2⫹ and Cd2⫹ clearly reduced theamplitude 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 theK(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

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

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 areshown for GT1-7 cells incubated with 300 pM E2 (E, n ⫽ 10) andwithout 300 pM E2 (F, n ⫽ 10). The data are repeated from Fig. 1B.
B, Concentration-response relationship of E2 on the Ca2⫹-insensitiveK⫹ currents at ⫹60 mV (n ⫽ 7–10).

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 notedthat the currents from the control cells were not increased at
potentials more positive than ⫹20 mV. In cells incubatedwith 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 incontrol 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 withE2 (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, IAHP
were hardly observed in cells incubated with or without 300
pm E2 (Fig. 4C).
In addition to the K(Ca) currents, the voltage-gated Ca2⫹
currents were examined in the presence of 10 mm Ca2⫹ in theextracellular solution. The maximal amplitude was observedat around ⫹20 mV in GT1-7 cells (Fig. 5A). Ca2⫹ currents at⫹20 mV were ⫺19.3 ⫾ 3.0 pA/pF in control cells and ⫺25.9 ⫾4.3 pA/pF in cells incubated with 300 pm E2 for 3 d (Fig. 5B).
There was no statistically significant effect of E2 on the Ca2⫹
currents. Simultaneous application of Ni2⫹ (100 ␮m) and

Endocrinology, February 2008, 149(2):774 –782

777

FIG. 3. Effect of E2 on the K(Ca) currents. The K(Ca) currents wereobtained by subtracting the Ni2⫹- and Cd2⫹-insensitive currents fromthe total K⫹ currents. Cells were incubated with E2 for 3 d. A, Current-voltage relationships of the K(Ca) currents in the control cells (F,n ⫽ 10) and in the cells incubated with 300 pM E2 for 3 d (E, 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 tobe 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 thecurrent 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.

Cd2⫹ (200 ␮m) completely blocked the Ca2⫹ currents (Fig. 5,
A and B). Pharmacological dissection with specific blockers
determined the diversity of voltage-gated Ca2⫹ channel subtypes in GT1-7 cells. The R-type Ca2⫹ channel blocker SNX486 (100 nm), the L-type Ca2⫹ channel blocker nifedipine (10␮m), and the T-type Ca2⫹ channel blocker Ni2⫹ (100 ␮m) wereused. SNX-486, nifedipine, and Ni2⫹ inhibited the Ca2⫹ currents by 72.2 ⫾ 3.6, 10.2 ⫾ 2.3, and 3.3 ⫾ 1.1%, respectively,in control cells (n ⫽ 10), and by 77.5 ⫾ 2.2, 8.5 ⫾ 1.7, and 4.9 ⫾
1.4%, respectively, in cells incubated with 300 pm E2 for 3 d(n ⫽ 10).
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 ␮m (Fig. 6). To
examine whether E2 affected either ER␣ or ER␤, a selective
agonist for each subtype of ER was used. The selective agonist for ER␤ (DPN) augmented the K(Ca) currents, whereasthat for ER␣ (PPT) had no effect on K(Ca) currents (Fig. 7A).

778

Endocrinology, February 2008, 149(2):774 –782

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

FIG. 5. Voltage-gated Ca2⫹ currents. Ca2⫹ currents were activatedby 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) andthose recorded from E2-treated cells (300 pM; n ⫽ 10). Ni2⫹ and Cd2⫹
blocked the Ca2⫹ currents in the both cell groups (E).

Knockdown of ER␣ and ER␤

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 evokedby voltage steps from ⫺60 to ⫹60 mV in 10-mV increments from aholding potential of ⫺90 mV. Data for Ni2⫹ and Cd2⫹ are not shownfor clarity. B, ChTX (100 nM)-sensitive currents (black bars), Ni2⫹ (100␮M)- and Cd2⫹ (200 ␮M)-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, Voltageprotocol and example traces for IAHP. The currents were activated bya 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.

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 noeffect on K(Ca) currents (Fig. 7, A and B).

ER␣ and ER␤ transcripts were successfully knocked downin GT1-7 cells (Fig. 8A). Preincubation with siRNA targeted
to ER␤ reduced the K(Ca) currents in GT1-7 cells incubatedwith 300 pm E2 for 3 d (Fig. 8B). By contrast, siRNA targeted
to ER␣ failed to reduce the K(Ca) currents (Fig. 8B). Significant decreases in the K(Ca) currents in the ER␤ knockdown(ER␤-KD) cells were observed at ⫹40 to ⫹60 mV comparedwith control cells, whereas the K(Ca) currents were not reduced in ER␣-KD cells (Fig. 8B).
Expression of BK channel mRNAs

To examine the expression levels of BK channel subunits,
RT-PCR analysis was performed on GT1-7 cells (Fig. 9). RTPCR revealed the expression levels of four different BK channel subunits in GT1-7 cells: ␣-, ␤1-, ␤2-, and ␤4-subunits (Fig.
9A). The amplicon length for the ␣-subunit in the GT1-7 cells
was 81 bp shorter than that in the mouse hypothalamus (26)(Fig. 9A), indicating the presence of a splice variant lacking
the 23rd exon (27) in GT1-7 cells. We failed to detect the␤3-subunit in GT1-7 cells (Fig. 9A). As negative controls,
PCRs were performed without cDNA (⫺ in Fig. 9A). Semiquantitative analysis of the PCR products revealed that thelevels of mRNAs for the ␣- and ␤4-subunits were significantly increased by incubating cells with 300 pm E2 for 3 d(Fig. 9B). However, E2 had no effect on the levels of mRNAs
for the ␤1- and ␤2-subunits (Fig. 9B).

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

FIG. 6. The ER antagonist ICI blocked the E2-induced augmentationof the K(Ca) currents. A, Current-voltage relationships of the K(Ca)currents. The augmentation by 300 pM E2 (F, n ⫽ 10) was completely
blocked by 3 ␮M ICI (E, n ⫽ 10). The data with 300 pM E2 alone arerepeated from Fig. 3A. B, The concentration-response relationship of
ICI in the presence of 300 pM E2 is shown at a voltage-pulse of ⫹60mV (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.

Endocrinology, February 2008, 149(2):774 –782

779

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(E, n ⫽ 10), whereas the ER␤-selective agonist DPN increased the BK
currents at ⫹30 mV, ⫹50 mV and ⫹60 mV (‚, n ⫽ 10). The control
data (F, 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.

Discussion

The present study revealed that E2 positively modulated
K(Ca) currents via ER␤ in GT1-7 cells. The involvement ofER␤ in the augmentation of K(Ca) currents was clearly demonstrated by the following observations: 1) an ER antagonist
completely blocked the response to E2 (Fig. 6); 2) a selective
agonist for ER␤, but not one for ER␣, mimicked this response(Fig. 7); and 3) knockdown of ER␤, but not of ER␣, blocked
the response (Fig. 8).
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-cellcurrents induced by depolarizing voltage pulses has beenreported (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 theexperimental protocol. The IK currents, like the SK currents,
are voltage independent and solely activated by increases inCa2⫹ 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 influxof 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 ofcurrents at potentials more positive than ⫹20 mV are carriedthrough BK channels, because the BK channels are dually

780

Endocrinology, February 2008, 149(2):774 –782

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

FIG. 8. Effect of ER knockdown on K(Ca) currents. Both ER␣ andER␤ were knocked down by siRNA. Control cells were transfected
only with pCAGIPuro-EGFP, encoding enhanced green fluorescentprotein. All transfected cells were cultured with 300 pM E2. A, Thelevels of mRNAs for the ERs were measured using RT-PCR in control(Cont.) and siRNA-treated cell groups targeted at ER␣ (ER␣-KD) andER␤ (ER␤-KD). B, Current-voltage relationships of the K(Ca) currents. The facilitatory effect of E2 was not observed in ER␤-KD cells(‚, n ⫽ 7). However, augmentation by E2 of the K(Ca) currents wasobserved in both ER␣-KD (E, n ⫽ 7) and control cells (F, n ⫽ 7). *, P ⬍0.05 vs. control; **, P ⬍ 0.01 vs. control using the Tukey-Kramer
multiple comparison test.

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 causea small increase in the activation of BK channels at thesepotentials. The conductance values, calculated by using thevalue 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 thefunctional expression of BK channels. Alternatively, thefunction 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 ␤-subunits (36, 37). To date, one type of ␣-subunit and four types of ␤-subunit (␤1–␤4) have been identified, different combinations of which form functional BK
channels with different characteristics (38 – 42). GT1-7 cells
expressed ␣-, ␤1-, ␤2-, and ␤4-subunits, similar to the expression pattern seen in the mouse hypothalamus (Fig. 9A)
and rat GnRH neurons (our unpublished data). The ␤3-subunit, which is expressed mainly in testis, pancreas, andspleen (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 ␤4-subunit
in GT1-7 cells, because coexpression of the ␤4-subunit with

FIG. 9. RT-PCR analysis of the expression of BK channel ␣- and ␤1to ␤4-subunit mRNAs in GT1-7 cells. Mouse hypothalamus and testis
were used as positive controls. PCRs were performed with (⫹) orwithout (⫺) cDNA. B, The levels of mRNAs for ␣- and ␤4-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 ␤1- and ␤2-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.

the ␣-subunit in Xenopus oocytes reduces sensitivity to blockade by ChTX (40). Interestingly, the mRNA and proteins forthe ␤1-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 ␤4-subunits,
without affecting those for the ␤1- and ␤2-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 ␤4-subunit probably attenuates thesensitivity of the channel to ChTX. These transcriptional
regulations of BK channels by E2 are likely to be at least inpart 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

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells

secretion by rat pituitary cells (45) and uptake of dopamine
into fetal rat hypothalamic cells (46).
In contrast to previous reports that demonstrated acuteeffects of E2 on K⫹ currents (3–5), including BK currents (8,9), we observed only a long-term effect (3 d) of E2. Thisdifference 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, underphysiological 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 ER␤, atleast 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 thepossibility that expression of BK channels in GnRH neurons
is directly regulated by E2 via ER␤, because GnRH neurons
express ER␤ (47– 49).
Acknowledgments
We are grateful to Drs. Yoshie Hiraizumi, Nobuyuki Tanaka, andChengzhu Yin and Ms. Sumiko Usui for their technical support.
Received June 8, 2007. Accepted October 16, 2007.
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@nms.ac.jp.
This research was supported in part by JSPS Grants-in-Aid for Scientific Research (16590180, 18390070, and 18590226), a MEXT Grant-inAid for Scientific Research on Priority Areas (1686210), and an Ishidsu
Shun Memorial Scholarship.
Disclosure Statement: The authors have nothing to disclose.

References
1. Petersen SL, Ottem EN, Carpenter CD 2003 Direct and indirect regulation of
gonadotropin-releasing hormone neurons by estradiol. Biol Reprod 69:1771–
1778
2. Watson CS, Gametchu B 2003 Proteins of multiple classes may participate innongenomic steroid actions. Exp Biol Med (Maywood) 228:1272–1281
3. Nabekura J, Oomura Y, Minami T, Mizuno Y, Fukuda A 1986 Mechanism of
the rapid effect of 17 ␤-estradiol on medial amygdala neurons. Science 233:
226 –228
4. Kelly MJ, Ronnekleiv OK, Eskay RL 1984 Identification of estrogen-responsive LHRH neurons in the guinea pig hypothalamus. Brain Res Bull 12:399 – 407
5. Minami T, Oomura Y, Nabekura J, Fukuda A 1990 17␤-Estradiol depolarization of hypothalamic neurons is mediated by cyclic AMP. Brain Res 519:
301–307
6. Abe H, Terasawa E 2005 Firing pattern and rapid modulation of activity by
estrogen in primate luteinizing hormone releasing hormone-1 neurons. Endocrinology 146:4312– 4320
7. Kow LM, Devidze N, Pataky S, Shibuya I, Pfaff DW 2006 Acute estradiol
application increases inward and decreases outward whole-cell currents ofneurons in rat hypothalamic ventromedial nucleus. Brain Res 1116:1–11
8. White RE, Han G, Maunz M, Dimitropoulou C, El-Mowafy AM, Barlow RS,
Catravas JD, Snead C, Carrier GO, Zhu S, Yu X 2002 Endothelium-independent effect of estrogen on Ca2⫹-activated K⫹ channels in human coronary
artery smooth muscle cells. Cardiovasc Res 53:650 – 661
9. Valverde MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI, Mann
GE, Vergara C, Latorre R 1999 Acute activation of Maxi-K channels (hSlo) by
estradiol binding to the ␤ subunit. Science 285:1929 –1931

Endocrinology, February 2008, 149(2):774 –782

781

10. Smith MS, Freeman ME, Neill JD 1975 The control of progesterone secretion
during the estrous cycle and early pseudopregnancy in the rat: prolactin,
gonadotropin and steroid levels associated with rescue of the corpus luteumof pseudopregnancy. Endocrinology 96:219 –226
11. Bergman MD, Schachter BS, Karelus K, Combatsiaris EP, Garcia T, Nelson
JF 1992 Up-regulation of the uterine estrogen receptor and its messenger
ribonucleic acid during the mouse estrous cycle: the role of estradiol. Endocrinology 130:1923–1930
12. DeFazio RA, Moenter SM 2002 Estradiol feedback alters potassium currents
and firing properties of gonadotropin-releasing hormone neurons. Mol Endocrinol 16:2255–2265
13. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI 1990
Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10
14. Charles AC, Hales TG 1995 Mechanisms of spontaneous calcium oscillations
and action potentials in immortalized hypothalamic (GT1-7) neurons. J Neurophysiol 73:56 – 64
15. Hales TG, Sanderson MJ, Charles AC 1994 GABA has excitatory actions on
GnRH-secreting immortalized hypothalamic (GT1-7) neurons. Neuroendocrinology 59:297–308
16. Martinez de la Escalera G, Choi AL, Weiner RI 1994 Biphasic GABAergic
regulation of GnRH secretion in GT1 cell lines. Neuroendocrinology 59:420 –425
17. Wetsel WC, Valenca MM, Merchenthaler I, Liposits Z, Lopez FJ, Weiner RI,
Mellon PL, Negro-Vilar A 1992 Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons. Proc
Natl Acad Sci USA 89:4149 – 4153
18. Bosma MM 1993 Ion channel properties and episodic activity in isolated
immortalized gonadotropin-releasing hormone (GnRH) neurons. J Membr
Biol 136:85–96
19. Spergel DJ, Catt KJ, Rojas E 1996 Immortalized GnRH neurons express largeconductance calcium-activated potassium channels. Neuroendocrinology 63:
101–111
20. Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS 1999 Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion. Proc Natl Acad Sci USA 96:4101– 4106
21. Navarro CE, Saeed SA, Murdock C, Martinez-Fuentes AJ, Arora KK, Krsmanovic LZ, Catt KJ 2003 Regulation of cyclic adenosine 3⬘,5⬘- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane
estrogen receptors in immortalized gonadotrophin-releasing hormone neurons. Mol Endocrinol 17:1792–1804
22. Roy D, Angelini NL, Belsham DD 1999 Estrogen directly represses gonadotropin-releasing hormone (GnRH) gene expression in estrogen receptor-␣(ER␣)- and ER␤-expressing GT1-7 GnRH neurons. Endocrinology 140:5045–
5053
23. Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A,
Ueda R, Saigo K 2004 Guidelines for the selection of highly effective siRNAsequences for mammalian and chick RNA interference. Nucleic Acids Res
32:936 –948
24. Kato M, Ui-Tei K, Watanabe M, Sakuma Y 2003 Characterization of voltagegated calcium currents in gonadotropin-releasing hormone neurons taggedwith green fluorescent protein in rats. Endocrinology 144:5118 –5125
25. Watanabe M, Sakuma Y, Kato M 2004 High expression of the R-type voltagegated Ca2⫹ channel and its involvement in Ca2⫹-dependent gonadotropinreleasing hormone release in GT1-7 cells. Endocrinology 145:2375–2383
26. Pallanck L, Ganetzky B 1994 Cloning and characterization of human andmouse homologs of the Drosophila calcium-activated potassium channel gene,slowpoke. Hum Mol Genet 3:1239 –1243
27. Nehrke K, Quinn CC, Begenisich T 2003 Molecular identification of Ca2⫹activated K⫹ channels in parotid acinar cells. Am J Physiol Cell Physiol 284:
C535–C546
28. Sah P 1996 Ca2⫹-activated K⫹ currents in neurones: types, physiological roles
and modulation. Trends Neurosci 19:150 –154
29. Maylie J, Bond CT, Herson PS, Lee WS, Adelman JP 2004 Small conductanceCa2⫹-activated K⫹ channels and calmodulin. J Physiol 554:255–261
30. Sailer CA, Hu H, Kaufmann WA, Trieb M, Schwarzer C, Storm JF, Knaus
HG 2002 Regional differences in distribution and functional expression ofsmall-conductance Ca2⫹-activated K⫹ channels in rat brain. J Neurosci 22:
9698 –9707
31. Kato M, Tanaka N, Usui S, Sakuma Y 2006 The SK channel blocker apamin
inhibits slow afterhyperpolarization currents in rat gonadotropin-releasing
hormone neurones. J Physiol 574:431– 442
32. Spergel DJ 2007 Calcium and small-conductance calcium-activated potassiumchannels in gonadotropin-releasing hormone neurons before, during, and after
puberty. Endocrinology 148:2383–2390
33. Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, Maylie J 1997 A
human intermediate conductance calcium-activated potassium channel. Proc
Natl Acad Sci USA 94:11651–11656
34. Pallotta BS, Magleby KL, Barrett JN 1981 Single channel recordings of Ca2⫹activated K⫹ currents in rat muscle cell culture. Nature 293:471– 474
35. Cui J, Cox DH, Aldrich RW 1997 Intrinsic voltage dependence and Ca2⫹

782

Endocrinology, February 2008, 149(2):774 –782

regulation of mslo large conductance Ca-activated K⫹ channels. J Gen Physiol
109:647– 673
36. Knaus HG, Garcia-Calvo M, Kaczorowski GJ, Garcia ML 1994 Subunit composition of the high conductance calcium-activated potassium channel fromsmooth muscle, a representative of the mSlo and slowpoke family of potassiumchannels. J Biol Chem 269:3921–3924
37. McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ
1995 Functional role of the ␤ subunit of high conductance calcium-activatedpotassium channels. Neuron 14:645– 650
38. Brenner R, Jegla TJ, Wickenden A, Liu Y, Aldrich RW 2000 Cloning andfunctional characterization of novel large conductance calcium-activated potassium channel ␤ subunits, hKCNMB3 and hKCNMB4. J Biol Chem 275:
6453– 6461
39. Cox DH, Aldrich RW 2000 Role of the ␤ subunit in large-conductance Ca2⫹activated K⫹ channel gating energetics. Mechanisms of enhanced Ca2⫹ sensitivity. J Gen Physiol 116:411– 432
40. Meera P, Wallner M, Toro L 2000 A neuronal ␤ subunit (KCNMB4) makes thelarge conductance, voltage- and Ca2⫹-activated K⫹ channel resistant tocharybdotoxin and iberiotoxin. Proc Natl Acad Sci USA 97:5562–5567
41. Xia XM, Ding JP, Lingle CJ 1999 Molecular basis for the inactivation of Ca2⫹and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells. J Neurosci 19:5255–5264
42. Xia XM, Ding JP, Zeng XH, Duan KL, Lingle CJ 2000 Rectification and rapid
activation at low Ca2⫹ of Ca2⫹-activated, voltage-dependent BK currents:

Nishimura et al. • E2 Augments K(Ca) Currents in GT1-7 Cells
consequences of rapid inactivation by a novel ␤ subunit. J Neurosci 20:4890 –4903
43. Benkusky NA, Korovkina VP, Brainard AM, England SK 2002 Myometrial
maxi-K channel ␤1 subunit modulation during pregnancy and after 17␤estradiol stimulation. FEBS Lett 524:97–102
44. Nagar D, Liu XT, Rosenfeld CR 2005 Estrogen regulates ␤1-subunit expression
in Ca2⫹-activated K⫹ channels in arteries from reproductive tissues. Am J
Physiol Heart Circ Physiol 289:H1417–H1427
45. Emons G, Ortmann O, Thiessen S, Knuppen R 1986 Effects of estradiol andsome antiestrogens (clomiphene, tamoxifen, and hydroxytamoxifen) on luteinizing hormone secretion by rat pituitary cells in culture. Arch Gynecol
237:199 –211
46. Christian M, Gillies G 1999 Developing hypothalamic dopaminergic neurones as potential targets for environmental estrogens. J Endocrinol 160:R1–R6
47. Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD,
Liposits Z, Petersen SL 2000 Detection of estrogen receptor-␤ messenger
ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 141:3506 –3509
48. Hrabovszky E, Steinhauser A, Barabas K, Shughrue PJ, Petersen SL, Merchenthaler I, Liposits Z 2001 Estrogen receptor-␤ immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology
142:3261–3264
49. Kallo I, Butler JA, Barkovics-Kallo M, Goubillon ML, Coen CW 2001 Oestrogen receptor ␤-immunoreactivity in gonadotropin releasing hormone-expressing neurones: regulation by oestrogen. J Neuroendocrinol 13:741–748

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.