and Ca2+- Activated K+ - J-Stage

J. Physiol. Sci. Vol. 58, No. 1; Feb. 2008; pp. 21–29 Online Jan. 8, 2008; doi:10.2170/physiolsci.RP013207

REGULAR PAPER

Rat GnRH Neurons Exhibit Large Conductance Voltage- and Ca2+Activated K+ (BK) Currents and Express BK Channel mRNAs Yoshie HIRAIZUMI1,2, Ichiro NISHIMURA1, Hirotaka ISHII1, Nobuyuki TANAKA1, Toshiyuki TAKESHITA2, Yasuo SAKUMA1, and Masakatsu KATO1 1Department of Physiology and 2Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, 113-8602 Japan

Abstract: Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. As in other neurons, the discharge pattern of action potentials is important for these neurons to function properly. Therefore it is important to elucidate the expression patterns of various types of ion channels in these neurons because they determine cell excitability. To date, voltage-gated Ca2+ channels and SK channels have been reported to be expressed in rat GnRH neurons. In this study, we focused on K+ channels and analyzed their expression in primary cultured GnRH neurons, prepared from GnRH-EGFP transgenic rats, by means of perfo-

rated patch-clamp recordings. GnRH neurons exhibited delayed-rectifier K+ currents and large conductance voltage- and Ca2+-activated K+ (BK) currents. Moreover, multicell RT-PCR (reverse transcriptase–polymerase chain reaction) experiments revealed the expression of BK channel mRNAs (α, β1, β2, and β4). The results show the presence of delayed-rectifier K+ currents and BK currents besides previously reported slow afterhyperpolarization currents. These currents control the action potential repolarization and probably also the firing pattern, thereby regulating the cell excitability of GnRH neurons.

Key words: potassium channel, calcium-activated potassium channel, charybdotoxin, patch-clamp, RT-PCR.

Gonadotropin-releasing hormone (GnRH) neurons play

an essential role in the neuroendocrine control of reproduction. It is well established that they must fire action potentials to release GnRH in the median eminence, which regulates gonadotrophs in the anterior pituitary. Therefore it is important to elucidate the expression patterns of various types of ion channels in these neurons because they determine cell excitability [1]. Current-clamp experiments have shown K+ and Ca2+ channels to be expressed in mouse GnRH neurons [2]. Voltage-gated Ca2+ channels have been reported to be expressed in rat [3] and mouse GnRH neurons alike [4]. SK channels, which underlie slow afterhyperpolarization, have also been reported to be expressed in rat and mouse GnRH neurons [5, 6], and the presence of spike-dependent depolarizing afterpotentials has been demonstrated in mouse GnRH neurons [7]. In the present experiments, we study voltage-gated K+ channels and Ca2+-activated K+ [K(Ca)] channels, because these are also closely related to cell excitability [8, 9]. Three types of K(Ca) channels are known, namely, SK channels, IK channels, and BK channels [1, 10, 11]. The expression of SK channels has been reported in rat GnRH neurons [5]. IK channels have not been reported in normal brain tissue [12, 13] but they have been reported in peripheral tissues, such as pancreas, lung, prostate, thyroid, stomach,

bladder, and colon [12]. Thus it is unlikely that GnRH neurons express the IK channels. The focus in the present experiments is therefore to investigate whether or not rat GnRH neurons express BK channels. A delayed-rectifier K+ current is ubiquitously expressed in excitable cells [1], including GnRH neurons [2], and mainly contributes to action potential repolarization [1]. BK channels are expressed in various cell types [14, 15]. In neurons, they are reported to be involved in action potential repolarization and the fast afterhyperpolarization [10]. BK channels have a large single-channel conductance (100–250 pS) and are dually activated by membrane depolarization and increases in intracellular Ca2+ concentration ([Ca2+]i) [16]. These channels require submicromolar to ~10 µM Ca2+ for activation at depolarized membrane potentials (–30 to +60 mV) [17]. BK channels are formed of 4 α subunits and probably also 4 auxiliary β subunits [18]. The gating kinetics, Ca2+ sensitivity, and pharmacology are changed by the association of the 4 α subunits with different β subunits [19]. The BK β1-β4 subunits have been cloned [20–23], among which the β3 subunit is hardly detected in the brain, but it is expressed in the testis, pancreas, and spleen [24]. In this study, we investigated K+ currents in rat GnRH neurons by means of perforated patch-clamp experiments

Received on Nov 6, 2007; accepted on Jan 5, 2008; released online on Jan 8, 2008; doi:10.2170/physiolsci.RP013207 Correspondence should be addressed to: Masakatsu Kato, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo-ku, Tokyo, 113-8602 Japan. Fax: +81 3 5685 3055; E-mail: [email protected]

The Journal of Physiological Sciences Vol. 58, No. 1, 2008

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Y. HIRAIZUMI et al.

and revealed the presence of delayed-rectifier K+ currents and BK currents in these neurons. The BK currents showed low sensitivity to the BK channel blocker charybdotoxin (ChTX). Further, RT-PCR (reverse transcriptasepolymerase chain reaction) experiments revealed the expression of mRNAs encoding BK channels (α, β1, β2, and β4), but the mRNA encoding the β3 subunit was not detected. METHODS

All experiments were performed with the approval of the Nippon Medical School Animal Care Committee. Rats expressing enhanced green fluorescent protein (EGFP) under the control of the GnRH promoter [3] were used in these studies. The rats had free access to water and rat chow and were kept under a 14-h light, 10-h dark cycle. The estrous cycle was monitored by means of vaginal smear histology. Rats aged 2–3 months were used. Short-term dissociated culture. Brains were excised from rats under ether anesthesia in the afternoon (~3:00 PM). The medial septum, diagonal band of Broca (DBB), organum vasculosum of the lamina terminalis (OVLT), and medial preoptic area (mPOA) were cut out with a razor and surgical blades. The sections were minced and treated with papain (21 U/ml; Funakoshi, Tokyo, Japan) containing DMEM (Sigma, St. Louis, MO) for 50 min at 30°C. The tissues were triturated with a 5-ml plastic pipette after several washes with MEM (Invitrogen, Grand Island, NY). Cell suspensions were applied to a discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) density gradient composed of 1.0, 1.023, and 1.078 g/ml layers and centrifuged. The cells obtained from the middle layer were plated on polylysine-coated coverslips and cultured overnight in Neurobasal-A medium (Invitrogen) supplemented with 0.5 mM L-glutamine and B-27 (Invitrogen) at 37°C in 95% air and 5% CO2 in 35 mm dishes. The cells were used within 24 h. Most of the GnRH neurons were round, but some were spindleshaped. In some experiments, female data were grouped according to the estrous cycle stage, which was determined on the day of sacrifice. Electrophysiology. A List EPC-9 patch-clamp system (HEKA Electronik, Lambrecht/Pfalz, Germany) was used for the recordings and data analyses. Whole cell currents were recorded by means of the perforated patch-clamp technique with amphotericin B (Seikagaku Corp., Tokyo, Japan; Kato et al., 2003) at room temperature (25°C). The perforated patch-clamp configuration leaves the cell interior nearly intact, thereby blocking the rundown of the Ca2+ currents. This is critical because the Ca2+-activated K+ currents were examined in the present experiment. The final concentration of amphotericin B in the pipette solution was 0.05 mg/ml. The pipette solution consisted of 95 mM potassium aspartate, 47.5 mM KCl, 1 mM MgCl2, 0.1 22

mM EGTA, and 10 mM HEPES (pH 7.2), the osmolarity being adjusted to 270 mOsm. The extracellular solution consisted of 137.5 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 0.8 mM MgCl2, 0.6 mM NaHCO3, 10 mM glucose, and 20 mM HEPES (pH 7.4), the osmolarity being adjusted to 300 mOsm. To minimize the nonspecific binding of peptides, 0.01% cytochrome C (Wako Junyaku, Osaka, Japan) was included in the extracellular solution. The extracellular solution was not oxygenated. When the K+ current was recorded, 0.3 µM TTX (Seikagaku Corp.) was included to block the Na+ current. Pipettes were fabricated from borosilicate glass capillaries and had a resistance of 6–8 MΩ. The pipettes were targeted to the GnRH neurons in the extracellular solution without cytochrome C. After a neuron had been touched, slight negative pressure was applied to the pipette, leading to a seal resistance of ~5 GΩ. Perforation with amphotericin B was achieved within 3–7 min after gigaseal formation. The currents were filtered at 2.3 kHz, digitized at 10 kHz, and recorded. The series resistance was electronically compensated by 70%. Data were taken when the series resistance was stable and less than 30 MΩ. The leak currents ranged from –5 pA to –42 pA at –90 mV. The cell capacitance was 13.3 ± 3.4 pF (mean ± SD, n = 120). Capacitative and leak currents were subtracted by means of the P/4 protocol. In current-clamp experiments, a few pico-amps were injected when necessary to keep the membrane potential around –70 mV. Action potentials were elicited by 1 ms current pulses of 200–400 pA. The following procedures were taken to isolate K(Ca) currents. First, control currents were obtained by applying 50 ms voltage pulses (–60 mV to +60 mV in 10 mV increments) from the holding potential of –90 mV. Second, the same cell was exposed to 200 µM Ni2+ and 500 µM Cd2+, and currents were then recorded by applying the same protocol used to obtain control currents. Third, the difference currents were obtained by subtracting the currents obtained in the presence of Ni2+ and Cd2+ from the control currents. These difference currents represent the K(Ca) currents. The K(Ca) currents activated at +60 mV are referred to as the BK currents because they were not inhibited by the SK channel blocker apamin, as shown in Fig. 1C. To isolate low-voltage-activated transient K+ current, 200-ms prepulse (–90 mV to –30 mV in 10 mV increments) with 200-ms test pulse (+60 mV) was applied from the holding potential of –100 mV at 0.1 Hz (Fig. 2). The extracellular solution contained Ni2+ (100 µM) and Cd2+ (200 µM) to block the voltage-gated Ca2+ channels, thereby suppressing the K(Ca) currents. The currents activated without prepulse were compared with those activated with prepulses of –50 mV to –30 mV. Multicell RT-PCR. Coronal slices (200 µm thick) containing the medial septum, DBB, OVLT, and medial preoptic area (mPOA) were prepared from adult male and fe-


BK Channels in Rat GnRH Neurons Table 1. Primer sequences and PCR conditions. Gene name Kcnma1

Direction 1st PCR

(α) 2nd PCR Kcnmb1

1st PCR

(β1) 2nd PCR Kcnmb2

1st PCR

(β2) 2nd PCR Kcnmb3*

1st PCR

(β3) 2nd PCR Kcnmb4

1st PCR

(β4) 2nd PCR GnRH

Primer sequence (5' to 3')

forward

5'-GCGGGGGCAAGACGAAGGA-3'

reverse

5'-AAACCGCAAGCCAAAGTAGAGAAG-3'

forward

5'-GAAGGAGGCCCAGAAGATAAACAA-3'

reverse

5'-CCGCAAGCCAAAGTAGAGAAGGAA-3'

forward

5'-GTGCCGCCATCACTTACTACAT-3'

reverse

5'-TCTGCCCACAGCTGATACATTG-3'

forward

5'-TACCACTGTGCTGCCCCTCTACCA-3'

reverse

5'-TGCCCACAGCTGATACATTGACC-3'

forward

5'-TTCCTACTGGGAATCACACTGCT-3'

reverse

5'-TGACGCTCTTTTGGTTCCCTTCTG-3'

forward

5'-GTGTGCCCTGCTGAATGTGTC-3'

reverse

5'-GAATAGCAGGGGAAGTGTTGGTGTCTC-3'

forward

5'-GGCCTCCTCCGTGCTGATGTTCTT-3'

reverse

5'-GCCAGCCCGCAGGATGTGTTCTA-3'

forward

5'-CTCCTCCGTGCTGATGTTCTTCCT-3'

reverse

5'-CGCCTCCCAGCAATGTCAGTAAA-3'

forward

5'-GGGGCACCTCGCAGTATCC-3'

reverse

5'-GGCTTCCCACAATCCTCCTC-3'

forward

5'-CTACACAGCGACCAGCACCAG-3'

reverse

5'-CCACCGGCCAGAGGAAGCAGT-3'

forward

5'-ACTGATGGCCGCTGTTGTTCT-3'

reverse

5'-CTTCTTCTGCCCAGCTTCCTCTTCA-3'

Product size (bp1)

Ta2 (C)

PCR cycles

338

60

24

322

60

24

182

58

24

152

62

24

370

56

25

279

61

25

578

63

30

464

60

30

428

58

26

238

58

26

256

58

38

1bp,

base pair; 2Ta, annealing temperature; *, the sequence for Kcnmb3 (β3) was newly determined and registered (accession number: AB297662). Forward and reverse primers were designed on different exons to distinguish the amplification of cDNA templates from that of contaminating genomic DNA. To design the PCR primers, we referred to the following nucleotide sequences: U40603 for α, U40602 for β1, AJ517198 for β2, AB297662 for β3, AY028605 for β4, and M12579 for GnRH.

male rats. The animals were decapitated under ether anesthesia. Their brains were quickly removed and immersed in an ice-cold oxygenated (95% O2, 5% CO2) cutting solution consisting of 2.5 mM KCl, 1.25 mM Na2HPO4, 0.6 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, 10 mM HEPES, 7 mM glucose, 248 mM sucrose, 1.3 mM ascorbic acid, and 3 mM Na-pyruvate (pH 7.4, 290 mOsm). The brains were blocked, glued with cyanoacrylate to the chilled stage of a Vibratome VIB3000 (Vibratome, St. Louis, MO), and cut. The slices were then incubated at 30°C for 30 min in oxygenated artificial cerebrospinal fluid (ACSF) containing 137.5 mM NaCl, 2.5 mM KCl, 1.25 mM Na2HPO4, 0.6 mM NaHCO3, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4, 290 mOsm), and thereafter they were kept at room temperature. Each slice was transferred to the recording chamber, kept submerged, and continuously superfused with oxygenated ACSF at a rate of 3 ml/min. The slice was viewed under an upright fluorescence microscope (BX50; Olympus, Tokyo, Japan) using a 40× waterimmersion objective lens. Pipettes of 1–2 MΩ (inner tip

diameter, 4–6 µm) were fabricated from glass capillaries baked at 200°C for 5 h. Each pipette was filled with 5 µl of an autoclaved solution comprising 150 mM KCl, 3 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.2, 270 mOsm). Positive pressure was applied to the pipette, and the pipette was then targeted to GnRH neurons. After a cell was reached, positive pressure was removed and negative pressure was applied for the sealing and breaking of the patch membrane. The cytoplasmic contents were harvested under visual control from 5 GnRH neurons either in the DBB/OVLT region or in the mPOA and pooled in a thin-wall PCR tube containing RNase inhibitor (RNasin Plus; Promega, Madison, WI). The harvested contents were heated with random hexamer primers (Promega) at 95°C for 5 min and then cooled on ice for 1 min. The reverse transcription mixture (50 µl) contained cytoplasmic contents from 5 cells, 1× reverse transcription buffer, 1 mM dNTP mixture, 4 µg random hexamer primers, 40 U RNase inhibitor, and 200 U M-MLV RTase [RNase H (–); RevaTra Ace; Toyobo, Osaka, Japan]. Reverse transcription was carried


23

Y. HIRAIZUMI et al.

Ref

Water, +RT

No suction

+ RT

No suction

- RT

+ RT

Marker

- RT

mPOA

DBB/OVLT

D E1 E2 E3 E4 GnRH E-actin Fig. S1. Negative controls for RT-PCR amplification of BK channel subunits mRNA. A specific PCR amplification of BK channel subunits (α, β1-4), GnRH, and β-actin mRNAs was confirmed by using appropriate samples as negative controls. Three kinds of samples were prepared: (i) cytosols from GnRH neurons treated without reverse-transcriptase (–RT); (ii) samples without suction of cytosols (No suction); and (iii) the same amount of water was added to the PCR tube instead of suctioned cytosols (Water, +RT). In obtaining the “No suction” samples, GnRH neurons were touched by the pipette, but the cytosols were not sucked into the pipette. +RT, cytosols from GnRH neurons that were treated with reverse transcriptase. For "+RT" samples, we selected the positive ones, except for β3, and electrophoresed them. RT-PCR was performed as described in MATERIALS AND METHODS. PCR for β-actin was performed with 1 round of 40 cycles using the following primers: 5'-GTCCACACCCGCCACCAGT-3' (forward) and 5'-CGTCTCCGGAGTCCATCACAAT-3' (reverse). Four independent samples of negative controls yielded no bands (n = 4), indicating that the RT-PCRs performed in the present experiments successfully detected the specific expression of mRNA in GnRH neurons without contamination. Ref, reference tissues (hypothalamic tissues for α, β1, β2 and β4; testis for β3).

out at 30°C for 10 min, then at 42°C for 45 min. After stopping the reaction by heating at 75°C for 15 min, we treated the reaction mixture with RNase H (Takara BIO, Shiga, Japan) at 37°C for 30 min and stored it at –20°C until use. To confirm successful cDNA synthesis from the cytoplasmic contents of GnRH neurons, we performed a oneround PCR amplification of GnRH mRNA transcripts, using 5 µl of the reverse transcription mixture as template. For BK channel subunits (α and β1-4), a two-round PCR amplification was performed, using 20 µl of the reverse 24

transcription mixture as a template for the first PCR and 1 µl of the first PCR solution as a template for the second PCR. The PCR conditions were 94°C for 2 min, 24–30 cycles of 94°C for 30 s, 56º–63°C for 20 s, 72°C for 30 s, and 72°C for 5 min. The PCR mixture (50 µl) contained template DNA, 1× PCR buffer, 0.2 mM dNTP mixture, 0.2 µM forward and reverse primers, and 1.2 U Blend Taq (Toyobo). Primer sequences and detailed PCR conditions are shown in Table 1. The amplified products were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized under UV irradiation. Gel images were captured using the FAS-III system (Toyobo). The identities of PCR amplicons were confirmed by DNA sequencing. We also performed single-cell RT-PCR for GnRH mRNA transcripts and found that all 12 samples examined were positive (data not shown). This indicates that we successfully harvested cytosols from GnRH neurons, but not from non-GnRH neurons. Also, we carried the whole procedure without sucking the cystosols (no suction) or adding reverse transcriptase (–RT) and achieved negative results in both (supplemental data: Fig. S1). In the present experiments, the cytoplasmic contents were harvested from GnRH neurons in the slice preparations, but not from those in dispersed culture because the levels of the cellular mRNAs decrease during the dispersion and overnight culture. Chemicals. We obtained nifedipine and 4-aminopyridine (4AP) from Wako Junyaku. We purchased ω-conotoxin GVIA (GVIA), ω-agatoxin IVA (Aga-IVA), SNX482, and ChTX from the Peptide Institute (Osaka, Japan). Statistics. Data were obtained from at least two or three independent experiments. They are expressed as means ± SEM unless otherwise mentioned. The Tukey-Kramer multiple comparison test, the Wilcoxon signed rank test, and the paired t-test were used for statistical analyses. The significance level was set at P < 0.05. RESULTS

K currents in GnRH neurons

A cell was held at –90 mV, and the membrane potential was stepped to depolarizing voltages (–60 mV to +60 mV in 10 mV increments) for 50 ms at 0.2 Hz to activate K+ currents (Fig. 1A). The K+ currents were activated from – 30 mV and at more positive potentials (Fig. 1B). The initial value, which was determined as the charge density for the initial 5 ms of the voltage pulse, was 2.3 ± 0.3 pQ/pF (n = 8) at +60 mV in the control. The late value, which was determined as the mean current density for the last 2.5 ms of the voltage pulse, was 192 ± 21 pA/pF (n = 8) at +60 mV in the control. The BK channel blocker ChTX (1 µM; [25, 26]) attenuated the currents by 16.9 ± 2.4% (n = 8) in the initial value and 18.7 ± 3.9% (n = 8) in the late value (Fig. 1C). ChTX inhibited the currents by ~8% at 0.1 µM and by ~15% at 0.5 µM in the initial and also the late val-


BK Channels in Rat GnRH Neurons B -60 mV -90 mV

Control

ChTX (1 PM)

Ni2+, Cd2+

0.5 nA 10 ms

+60 mV (200 ms) Current density (pA/pF) Charge density (pQ/pF)

+60 mV (50 ms)

A

Initial value 2.5 2.0 1.5

-40 mV (200 ms)

Control ChTX Ni, Cd

-100 mV

1.0 0.5 0 200

Late value

0.5 nA

100

0 -60

0

+60

+60 mV

Test pulse K+ current at +60 mV (% of control)

C Initial Late

100

-30 mV -40 mV -50 mV

-100 mV

80 60 40 20

1 nA

0 0.1 0.1 Apamin (PM)

0.5 ChTX (PM)

1

Ni2+, Cd2+

Fig. 1. K+ currents in GnRH neurons. A: Cells were clamped at –90 mV and given 50-ms voltage pulses from –60 to +60 mV in 10 mV steps at 0.2 Hz, as shown at the top. Control currents, those with 1 µM ChTX and those with 200 µM Ni2+ and 500 µM Cd2+, are shown. B: The charge densities of the initial phase were determined for the current elicited by the initial 5 ms of the voltage pulse and were plotted against the test pulse potential. The mean current densities of the late phase were determined for the current during the last 2.5 ms of the voltage pulse and were plotted against the test pulse potential. C: Summary graph illustrating the effects of apamin (100 nM), ChTX (0.1–1 µM), and Ni2+ (200 µM) and Cd2+ (500 µM). The data were obtained from GnRH neurons isolated from adult males. The effects of the blockers were statistically significant in the Wilcoxon signed rank test (*P < 0.05 vs. control; **P < 0.01 vs. control, n = 8).

ues. However, the SK channel blocker apamin (100 nM; [27]) did not attenuate the currents, suggesting that the SK channels were not involved (Fig. 1C). The currents were further attenuated by a simultaneous application of 200 µM Ni2+ and 500 µM Cd2+, which completely block voltage-gated Ca2+ currents in GnRH neurons [3, 5], by 32.3 ± 1.8% (n = 8) in the initial value and by 33 ± 3.2% (n = 8) in the late value (Fig. 1C). The inhibitory effect of ChTX was not observed when Ni2+ and Cd2+ were applied prior

Fig. 2. Absence of low-voltage–activated transient current. The voltage protocols and representative current traces are shown. Traces in the upper panel are the currents without prepulse and those with prepulse of –40 mV. There was no apparent A current in this cell. In the lower panel, 4 traces are shown, and 3 of them are overlapped. The top trace is the current without prepulse, and the lower 3 traces are those with –50 to –30 mV. There were some suppressions of the current by the prepulse of –50 mV or more depolarized potentials. It is to be noted, however, that no transient current was activated by a prepulse of –50 to –30 mV (arrows). The data were obtained from GnRH neurons isolated from adult males and females.

to ChTX (data not shown). A washout of these blockers partially removed the inhibitions (data not shown). The presence of the low-voltage–activated transient K+ current was examined in Ni2+- and Cd2+-containing solution as shown in Fig. 2. The peak amplitude of the currents was determined by subtracting the current with a prepulse of –40 mV from that without prepulse. The peak amplitude of the currents was 10.6 ± 2.8 pA/pF (n = 10), and they ranged from 0 pA/pF to 22 pA/pF. However, these currents suppressed by prepulse decayed very little over the test pulse time of 200 ms. It is also to be noted that there was no apparent activation of the transient current by prepulses of –40 or even –30 mV (Fig. 2, arrows).


25

Y. HIRAIZUMI et al.

A

A

+60 mV (50 ms)

Control nifedipine

-90 mV Control

+GVIA, Aga-IVA, SNX-482 DRK

BK

1 nA 10 ms

B

0.5 nA

R

10 ms

L

B

BK initial BK late

C BK (% of total)

Current density (pA/pF)

DRK

100

40

20

P

E

males

Fig. 3. Delayed rectifier K+ currents and BK currents. A: The voltage protocol and representative current traces are shown. The application of Ni2+ and Cd2+ attenuated the currents. The remaining currents are referred to as the delayed-rectifier K+ (DRK) currents. The difference currents produced by subtracting the DRK currents from the control currents represent the Ni2+- and Cd2+-sensitive currents and are referred to as the large conductance voltage- and Ca2+-activated K+ (BK) currents. B: Summary graph showing DRK current densities and BK current densities. There was no statistically significant difference according either to sex or estrous cycle stage (n = 11–14) in the Tukey-Kramer multiple comparison test. D, diestrus; P, proestrus; E, estrus.

Delayed-rectifier K+ currents and BK currents

The application of Ni2+ and Cd2+ eliminated the Ca2+sensitive component of the K+ currents. These Ca2+-sensitive currents activated at +60 mV are referred to as BK currents (Fig. 3). Under these conditions, most of the remaining current is the delayed-rectifier K+ current because the low-voltage-activated transient K+ current was undetectable, as shown in Fig. 2. Therefore the currents insensitive to Ni2+ and Cd2+ are referred to as delayed-rectifier K+ currents. BK currents and delayed-rectifier K+ currents are shown collectively in Fig. 3B according to sex and estrous cycle stage. The mean values of BK currents were 98–125 pA/pF in the initial phase and 77–93 pA/pF in the late phase. The mean values of the late de26

D BK (% of total)

D

Initial value

males

0 0

D P E

60

L

N

P/Q

40

R

T residual Late value

20

0 L N P/Q R T residual Fig. 4. Effect of Ca2+ channel blockers on BK currents. Nifedipine (10 µM), GVIA (1 µM), Aga-IVA (200 nM), SNX-482 (100 nM), and Ni2+ (50 µM) were sequentially applied. A: The current was activated by a voltage pulse to +60 mV from the holding potential of –90 mV. Representative traces of the control, with nifedipine and with a combined application of nifedipine, GVIA, Aga-IVA, and SNX-482, are shown. Other current traces are not shown for clarity. B: Nifedipine-sensitive and SNX-482–sensitive components of BK currents are shown as L and R, respectively. C, D: The contribution of each type of Ca2+ channel to the generation of BK currents is shown. N, the current sensitive to GVIA; P/Q, the current sensitive to Aga-IVA; T, the current sensitive to Ni2+; residual, the current resistant to all of these blockers. The initial values are the charge densities for the initial 5 ms of the voltage pulse and are shown as a percentage of the total. The late values are the mean current densities during the last 2.5 ms of the voltage pulse. Data were taken from 11–14 cells. The comparison was made within either males or the same estrous cycle stage. *P < 0.05 vs. the other components in the TukeyKramer multiple comparison test.


BK Channels in Rat GnRH Neurons Ms

D

DBB/OVLT

mPOA

Nc Ref

F M F

E1 ChTX

1 ms

M F

E2

M

Fig. 5

Ni2+, Cd2+ F

Control Washout

E3

M F

Action potential duration (% of control)

E4 GnRH

200

F M

100

0 ChTX

Ni2+, Cd2+

Washout

Fig. 5. Effects of ChTX and Ni2+ and Cd2+ on the durations of action potentials. Action potentials were elicited by a 1 ms current pulse in the current-clamp mode. A: Action potential durations were determined at the half-amplitude of the action potential as indicated by the dotted line. ChTX (1 µM) lengthened the duration, and the following application of Ni2+ (200 µM) and Cd2+ (500 µM) further lengthened it. B: Summary graph illustrating the effects of channel blockers. *P < 0.05 vs. control (n = 5) in the paired t-test.

layed-rectifier K+ currents were 119–139 pA/pF. No statistically significant differences among these groups were noted in the Tukey-Kramer multiple comparison test. Effect of

M

Ca2+

channel blockers on BK currents

To analyze the relationship of Ca2+ channel subtypes with the generation of BK currents, the effects of subtypespecific Ca2+ channel blockers were examined. After the control current was recorded at the voltage-pulse of +60 mV, 10 µM nifedipine, 1 µM GVIA, 200 nM Aga-IVA, 100 nM SNX-482, and 50 µM Ni2+ were sequentially applied. The currents, which were inhibited by each blocker, were referred to as L-, N-, P/Q-, R-, and T-type–sensitive currents. The L- and R-type–sensitive components of the BK currents are presented in Fig. 4B. In the initial value, the R-type–sensitive component was ~43% of the total BK currents, and the L-type–sensitive one was ~20% (Fig.

Fig. 6. Multicell RT-PCR analysis of mRNAs encoding BK channel subunits in GnRH neurons. Cytosols, harvested from 5 GnRH neurons either in the DBB and OVLT regions (the first 5 lanes after the one showing the molecular size marker) or in the mPOA (the following 5 lanes), were pooled and reverse-transcribed to generate cDNA. The amount of cDNA corresponding to 2 cells was examined with each primer pair for α, β1-4, and GnRH. The gel shows the presence of the 322 bp α, 152 bp β1, 279 bp β2, 238 bp β4, and 256 bp GnRH amplicons. The amplicon for β3 was not detected in GnRH neurons. Ms, 100 bp ladder marker; Nc, without cDNA; Ref, reference tissues (hypothalamic tissue for α, β1, β2, β4, and GnRH; testis for β3); F, females; M, males.

4C). In the late value, the L-type–sensitive component was ~31%, and the others were less than ~20%, except for the N-type–sensitive component and the residual component during estrus (Fig. 4D). The residual component of the BK currents could be, at least in part, due to residual Ca2+ currents, because a simultaneous application of all of these Ca2+ channel blockers does not completely suppress Ca2+ currents [3]. There were no statistically significant differences according to sex or estrous cycle stage in the Tukey-Kramer multiple comparison test. Effects of ChTX and Ni2+ and Cd2+ on the duration of action potentials

Action potentials were generated by 1 ms current pulse (200–400 pA) in the current-clamp mode. The durations of the action potentials were 2.1 ± 0.1 ms (n = 5) at their half-amplitude in the control. The amplitude of the action potentials was determined as the difference between their threshold and peak potential. One µM ChTX increased the durations of action potentials by 16 ± 5%, and Ni2+ and Cd2+ further increased them by 54 ± 14% at the half-amplitude of the action potential (Fig. 5). A washout of the blockers for several minutes partially reversed these effects.


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Y. HIRAIZUMI et al. Transcripts for rat BK channels in GnRH neurons

To examine the expression of the mRNAs encoding BK channel subunits in GnRH neurons, RT-PCR experiments were performed (Fig. 6). Template cDNA corresponding to 2 cells was subjected to RT-PCR with specific primers for α and β1-4 subunits of the BK channels and for GnRH. A positive band for the α subunit appeared in all 10 reactions. One for the β1 subunit was seen in 6 of the 10 reactions performed, one for the β2 subunit in 2 of the 10 reactions performed, and one for the β4 subunit was seen in 2 or 3 of the 10 reactions performed. No positive bands were detected for the β3 subunit. GnRH was positive in all reactions. There was no clear difference in the expression pattern of the BK channel subunit mRNAs either between males and females or between the DBB/ OVLT and the POA. DISCUSSION

The results show the presence of delayed-rectifier K+ currents and BK currents in rat GnRH neurons. The low-voltage–activated current (A current) was not detected in rat GnRH neurons (Fig. 2). The activation threshold of the A current is ~–50 mV, and its half-activation potential is ~–20 mV [28]. If the A current is present, therefore, the transient currents must be activated by the pulses of –50 to –30 mV from the holding potential of –100 mV. No such transient currents were detected in the present experiments (Fig. 2, arrows), indicating very little presence of A current in the cell body of rat GnRH neurons. However, DeFazio and Moenter [29] reported the presence of the A current in mouse GnRH neurons. The different result might be due to the difference of the preparation. The present recordings were made in isolated rat GnRH neurons, whereas they performed the experiments in mouse GnRH neurons in slice preparation. The cause of the difference should be elucidated in future experiments. Both the delayed rectifier K+ currents and the BK currents were unaffected by either the sex or estrous cycle stage (Figs. 1 and 3). Three types of K(Ca) channels are known: SK, IK, and BK channels [1, 10, 11]. SK channels were not activated in the present experimental protocol (Fig. 1C), though they are expressed in rat GnRH neurons [5]. The SK channel blocker, apamin, had no effect on the K+ currents activated at +60 mV (Fig. 1). Moreover, we analyzed the membrane currents activated by the voltage pulse to +60 mV. At this potential, Ca2+-influx through the voltage-gated Ca2+ channels is small and, may not be enough to activate the SK channels. IK channels are unlikely to be expressed in rat GnRH neurons because they are not expressed in normal brain tissue [12, 13]. Therefore the K(Ca) currents activated at +60 mV in the present experiment are most likely to be mediated by BK channels. These channels are dually activated by membrane depolarization and increases in [Ca2+]i [16]. The blockade 28

of Ca2+ channels by Ni2+ and Cd2+ suppressed the K+ currents by ~30%, suggesting that ~30% of the depolarization-induced K+ currents are carried through BK channels in GnRH neurons (Fig. 1). These BK currents showed an initial peak followed by a gradual decline. The initial phase was most sensitive to the R-type Ca2+ channel blocker SNX-482 (Fig. 4), indicating that an influx of Ca2+ through the R-type channel is involved in generating the initial phase. This accords well with the finding that Rtype channels are rapidly activated and then follow a relatively fast inactivation [30]. The contribution of L-type channels is more than that of other channels in diestrus females and males. In the late phase, all 5 subtypes of Ca2+ channels equally contribute to the activation of BK channels. These findings are consistent with findings that Rtype Ca2+ currents contribute ~30% of the total voltagegated Ca2+ currents and that L-type currents contribute ~25% of them in adult GnRH neurons [3, 5]. Recently, BK channels affinity-purified from rat brain were shown to assemble into a macromolecular complex with the voltage-gated Ca2+ channels Cav1.2 (L-type), Cav2.1 (P/Qtype), and Cav2.2 (N-type), but not Cav2.3 (R-type) [31]. BK channels are often recognized by their high sensitivity to ChTX, but in the brain, many seem resistant to the usual nanomolar concentrations of the toxin. Toxin resistance can be caused by the β subunit. A coexpression of α and β1 subunits makes the typical toxin-sensitive channels. A coexpression of α and β4 subunits makes toxin-resistant channels [32]. It is suggested that the extracellular loop of the β4 subunit faces the pore of the BK channel and is very close to it, thereby interfering with toxin binding [32, 33]. The BK currents in rat GnRH neurons were relatively resistant to ChTX (Fig. 1). Moreover, GnRH neurons expressed mRNAs encoding α, β1, β2, and β4 subunits (Fig. 6). Therefore the low sensitivity to ChTX in GnRH neurons was caused, at least in part, by an expression of the β4 subunit. The GnRH neuronal cell line GT1-7 also exhibit BK currents [34, 35], and the expression pattern of BK channel subunit mRNA in these cells [35] is similar to that in rat GnRH neurons. To investigate the physiological role of the BK currents, we carried out a current-clamp experiment (Fig. 5). It is reported that BK currents contribute to a repolarization of action potentials and to the fast afterhyperpolarization [36]. In rat GnRH neurons, however, fast afterhyperpolarization current is not detected [5]. The duration of action potential at half-amplitude was lengthened by 1 µM ChTX and further lengthened by the complete blockade of Ca2+ channels by Ni2+ and Cd2+, suggesting that BK channels are involved in the repolarization of action potentials. In conclusion, rat GnRH neurons exhibit delayed-rectifier K+ currents and BK currents in addition to the SK currents [5]. These K+ currents are involved in the regulation of cell excitability and the firing pattern of action potentials. BK channels expressed in rat GnRH neurons are


BK Channels in Rat GnRH Neurons

formed of α subunits and auxiliary β1, β2, and β4 subunits. We are grateful to Drs. S. Sato, S. Yin, and Ms. S. Usui for their technical assistance. This research was supported in part by Grants-in-Aid for Scientific Research (16590180, 18390070, 18590226, 19790181) from the Japan Society for the Promotion of Sciences and a Grant-in-Aid for Scientific Research on Priority Areas (1686210) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. REFERENCES 1. Hille B. Ion channels of excitable membranes. Sunderland: Sinauer; 2001. 2. Sim JA, Skynner MJ, Herbison AE. Heterogeneity in the basic membrane properties of post natal gonadotropin-releasing hormone neurons in the mouse. J Neurosci. 2001;21:1067-75. 3. Kato M, Ui-Tei K, Watanabe M, Sakuma Y. Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats. Endocrinology. 2003;144:5118-25. 4. Nunemaker CS, DeFazio RA, Moenter SM. Calcium current subtypes in GnRH neurons. Biol. Reprod. 2003;69:1914-1922. 5. Kato M, Tanaka N, Usui S, Sakuma Y. The SK channel blocker apamin inhibits slow afterhyperpolarization currents in rat gonadotropin-releasing hormone neurones. J Physiol (London). 2006;574:431-42. 6. Spergel DJ. Calcium and small-conductance calcium-activated potassium channels in gonadotropin-releasing hormone neurons before, during, and after puberty. Endocrinology. 2007;148:2383-90. 7. Kuehl-Kovarik MC, Partin KM, Handa RJ, Dudek FE. Spike-dependent depolarizing afterpotentials contribute to endogenous bursting in gonadotropin releasing hormone neurons. Neuroscience. 2005;134:295-300. 8. Garcia ML, Hanner M, Knaus HG, Koch R, Schmalhofer W, Slaughter RS, Kaczorowski, GJ. Pharmacology of potassium channels. Adv Pharmacol. 1997;39:425-71. 9. Jan LY, Jan YN. Cloned potassium channels from eukaryotes and prokaryotes. Annu Rev Neurosci. 1997;20:91-123. 10. Sah P. Ca2+-activated K+ currents in neurones: types, physiological roles and modulation. Trends Neurosci. 1996;19:150-4. 11. Faber ES, Sah P. Calcium-activated potassium channels: multiple contributions to neuronal function. Neuroscientist. 2003;9:181-94. 12. Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci USA. 1997;94:11651-16. 13. Weaver AK, Bomben VC, Sontheimer H. Expression and function of calciumactivated potassium channels in human glioma cells. Glia. 2006;54:223-33. 14. Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P, Kaczorowski GJ, Garcia ML. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem. 1990;265:11083-90. 15. Latorre R, Oberhauser A, Labarca P, Alvarez O. Varieties of calcium-activated potassium channels. Annu Rev Physiol. 1989;51:385-99. 16. Pallotta BS, Magleby KL, Barrett JN. Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. Nature. 1981;29:471-4. 17. Cui J, Cox DH, Aldrich RW. Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels. J Gen Physiol. 1997;109:647-73. 18. Latorre R, Vergara C, Alvarez O, Stefani E, Toro L. Voltage-gated calciummodulated potassium cahnnels of large unitary conductance: structure, diversity,

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