Ca²⁺-Activated K⁺ Channels in Gonadotropin-Releasing Hormone-Stimulated Mouse Gonadotrophs

Abstract

GnRH receptor activation elicits release of intracellular Ca²⁺, which leads to secretion and also activates Ca²⁺-activated ion channels underlying membrane voltage changes. The predominant Ca²⁺-activated ion channels in rat and mouse gonadotrophs are Ca²⁺-activated K⁺ channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca²⁺] ([Ca²⁺]_i) and membrane current (I_m), and to identify specific Ca²⁺-activated K⁺ channels linking GnRH-induced increase in [Ca²⁺]_i to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways. Simultaneous measurement of [Ca²⁺]_i and I_m in voltage-clamped gonadotrophs revealed that GnRH stimulates an increase in [Ca²⁺]_i that precedes outward I_m, and that activates two kinetically distinct currents identified, using specific toxin inhibitors, as small conductance Ca²⁺-activated K⁺ (SK) current (I_SK) and large (big) conductance voltage- and Ca²⁺-activated K⁺ (BK) current (I_BK). We show that the apamin-sensitive current has an IC₅₀ of 69 pM, consistent with the SK2 channel subtype and confirmed by immunocytochemistry. The magnitude of the SK current response to GnRH was attenuated by 17β-estradiol (E₂) pretreatment. Iberiotoxin, an inhibitor of BK channels, completely blocked the residual apamin-insensitive outward I_m, substantiating that I_BK is a component of the GnRH-induced outward I_m. In contrast to its suppression of I_SK, E₂ pretreatment augmented peak I_BK. SK or BK channel inhibition modulated GnRH-stimulated LH secretion, implicating a role for these channels in gonadotroph function. In summary, in mouse gonadotrophs the GnRH-stimulated increase in [Ca²⁺]_i activates I_SK and I_BK, which are differentially regulated by E₂ and which may be targets for E₂ positive feedback in LH secretion.

The GnRH-stimulated rise in intracellular [Ca²⁺] activates both small and large conductance Ca2⁺-activated K⁺ currents, which are differentially regulated by estradiol-17β (E2) and which may be targets for E2 positive feedback in luteinizing hormone secretion.

In the anterior pituitary gland, the hypothalamic-releasing hormone GnRH has pleiotropic actions on gonadotrophs that span a time scale of minutes to hours. On the shortest time scale, GnRH elicits gonadotrophin secretion and characteristic changes in gonadotroph plasma membrane voltage (V_m). The link between gonadotroph GnRH receptor activation and electrical activity is the release of Ca²⁺ from intracellular stores that leads to hormone secretion and stimulation of plasma membrane Ca²⁺-activated ion channels that underlie the V_m changes (reviewed in Ref. 1). The predominant Ca²⁺-activated ion channels in rat and mouse gonadotrophs are Ca²⁺-activated K⁺ channels (1,2).

Activation of K⁺ channels hyperpolarizes the V_m. In rat gonadotrophs, hyperpolarizing oscillations in V_m track GnRH-induced oscillations in intracellular [Ca²⁺] ([Ca²⁺]_i) (3). V_m hyperpolarization removes voltage-dependent channel inactivation and, on subsequent depolarization, leads to the entry of extracellular (EC) Ca²⁺ through voltage-activated Ca²⁺ channels, which is necessary for Ca²⁺ homeostasis, including the maintenance of intracellular Ca²⁺ stores (1).

Several Ca²⁺-activated ion channels are present in the gonadotroph plasma membrane. Most studied are small conductance Ca²⁺-activated K⁺ (SK) channels, long known to be present in sheep (4) and rat gonadotrophs (5,6). In addition, large (big) conductance voltage- and Ca²⁺-activated K⁺ (BK) channels (7) and a Ca²⁺-activated nonspecific cation current (8) have been identified. Not as extensively studied as SK channels, the role of these channels in GnRH action involving Ca²⁺-dependent secretion and modulation of gonadotroph membrane excitability is not known.

SK channels, which are solely dependent on Ca²⁺ for activation, are fundamental components of cell excitability in, for example, neurons, smooth muscle cells, and secretory cells (9). Three genes encode the SK channel subunits (SK1, SK2, and SK3), and functional SK channels are complexes of four pore-forming subunits plus constitutively bound calmodulin that mediates Ca²⁺ gating of the channel (10,11). BK channels, which have dual dependence on V_m and Ca²⁺, are abundant, e.g. in brain, pancreas, and smooth muscle (12,13). Functional BK channels are complexes of tetrameric pore-forming α-subunits and auxiliary β-subunits, which can modify the Ca²⁺ sensitivity, inactivation, and pharmacological properties of the channels. Four genes encode for the β subunits (β1-β4), and the tissue-specific expression of these subunits is responsible for much of the functional diversity of BK channels.

Ca²⁺-activated ion channel regulation as a target for modulation of gonadotroph function has received relatively little attention. However, recently, we showed in female mouse gonadotrophs that 17β-estradiol (E₂) pretreatment of pituitary cell cultures decreases peak GnRH-induced membrane current (I_m) (2). This indicates an additional layer of functional complexity in the regulation of gonadotroph membrane activity.

The objectives were to establish the temporal relationship between GnRH-induced changes in [Ca²⁺]_i and I_m, and to identify specific Ca²⁺-activated K⁺ channel types in mouse gonadotrophs that link the GnRH-induced increase in [Ca²⁺]_i to changes in plasma membrane electrical activity and their modulation by E₂. The model was single gonadotrophs from adult female mice, and the electrophysiological approach was the perforated-patch configuration of the patch-clamp technique, which preserves signaling pathways, including GnRH-induced Ca²⁺ oscillations.

Materials and Methods

Materials

Media, trypsin, and sera were from Invitrogen-GIBCO (Carlsbad, CA). Kanamycin sulfate, BSA fraction V, amphotericin B, apamin, iberiotoxin, E₂, and GnRH were from Sigma-Aldrich Corp. (St. Louis, MO). Dow Corning R6101 was from K.R. Anderson (Morgan Hill, CA). Fura-2/AM and pluronic F-127 were from Invitrogen-Molecular Probes (Carlsbad, CA). For immunofluorescence, normal goat serum and mounting medium were from Vector Laboratories (Burlingame, CA), and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG and fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG were from Sigma-Aldrich. Monoclonal antibody 518B7 to bovine LH was provided by Dr. Jan Roser (University California, Davis, Davis, CA). SK2 polyclonal antibody to rat 542–559 antigen was from Alomone Labs (Jerusalem, Israel).

Animals and cell culture

The animal protocol was approved by the University of California Davis Institutional Animal Care and Use Committee. Adult female wild-type mice [C57/BL/6, Charles River (Hollister, CA)], in 12-h light, 12-h dark, were ovariectomized and maintained for 2 wk after surgery before use (2). Pituitary glands were removed after CO₂ narcosis and decapitation. Anterior pituitary tissue was enzymatically dispersed (trypsin) and prepared for cell culture as described (14).

Electrophysiology

Cells were plated in 35-mm dishes incorporating a 10 or 22-mm glass-bottomed well (World Precision Instruments, Sarasota, FL, or Bioscience Tools, San Diego, CA) at low density (∼15,000 cells per well) and flooded with MEM supplemented with 200 μm kanamycin sulfate, 10% fetal bovine serum that had been charcoal treated to remove endogenous steroids (15), and without (no steroid pretreatment) or with (E₂ pretreatment) 0.2 nm E₂. Cells were maintained in a humidified atmosphere (37 C) of 5% CO₂ in air and used on d 2–5 (day of plating = d 0).

Immunocytochemistry

Cells were plated on 12-mm glass coverslips (20,000 cells per coverslip) and cultured as described previously. On d 2, medium was replenished, and on d 3, cells were fixed for immunocytochemistry.

LH secretion

Cells were plated in 22-mm multi-well plates (2 × 10⁵ cells per well) and cultured as described previously with or without 0.2 nm E₂. Medium was replenished on d 2, and secretion experiments were performed on d 3.

Experimental procedures

Electrophysiology

Culture medium was replaced with an EC medium, without added E₂, consisting of (in mm): 135 NaCl, 5 KCl, 5 CaCl₂, 1.3 MgCl₂, 4 NaHCO₃, 5 glucose, 10 HEPES 1, mg ml⁻¹ BSA, and was adjusted to pH 7.4 with 5 n NaOH. Culture dishes were transferred to an inverted microscope with a stage maintained at 22 C and superfused with EC medium at approximately 1 ml min⁻¹. Gonadotrophs were tentatively identified based on morphological criteria as described (2). Putative gonadotrophs were voltage clamped at the indicated potential. Solution flow was switched between control EC medium and experimental media using a valve placed close to the microscope stage; GnRH, 1 nm, was applied for 1 min or as indicated. For toxin treatment, exposure was started 2 min before GnRH and was continued until the end of the GnRH treatment (or end of the acquisition). A I_m response to GnRH in voltage-clamped cells was taken as confirmation that the cell was a gonadotroph. Of 253 cells identified as putative gonadotrophs, 156 (62%) subsequently responded to GnRH, similar to our previously reported success rate, 58% (2). In all cases only data from GnRH-responsive cells are reported.

Measurements were performed using the perforated-patch configuration of the tight-seal patch-clamp recording technique (16). The pipette-filling solution consisted of (in mm): 130 KAspartate, 20 KCl, 10 NaCl, 5 MgSO₄, 10 HEPES, and was adjusted to pH 7.2 with 5 n KOH. Pipette fabrication and coating were as described (2). Perforation of the patch was achieved by inclusion of the pore-forming antibiotic amphotericin B. Series resistance compensation was not used; mean series resistance was 24 ± 0.5 MΩ (n = 144), and maximum uncorrected voltage error was less than 4 mV. Pipette potentials were corrected for junction and Donnan potentials, −16 mV, calculated as described (2). Recordings were made with a MultiClamp 700B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) via a DigiData 1320A computer interface (Molecular Devices) using Clampex (pClamp software suite; Molecular Devices). Data were filtered at 600 Hz and sampled at 50 kHz. GnRH-induced currents were recorded from cells voltage clamped at −50 or −66 mV. Reported currents were normalized to membrane capacitance (pA/pF) to correct for differences in cell size; there was no difference in membrane capacitance between control and E₂-pretreated cells [7.26 ± 0.13 pF (n = 73) vs. 7.09 ± 0.13 pF (n = 71); P = 0.341].

Simultaneous [Ca²⁺]_i and electrophysiology

[Ca²⁺]_i was determined ratiometrically with the fluorescent probe fura-2 as described (17,18) using hardware and software from Photon Technology International (Birmingham, NJ). Briefly, cells were loaded with the fluorophore by incubation with 2 μm fura-2/AM in serum-free MEM containing 1 mg ml⁻¹ BSA and without added steroids for 30 min at 37 C. The dishes were rinsed with EC medium and transferred to the microscope stage. Fluorescence, alternately excited at 340 and 380 nm, was collected using a 40 × CF Fluor oil-immersion lens and passed through a bandpass filter to a photomultiplier. [Ca²⁺]_i was calculated every 100 msec. Simultaneous recordings of [Ca²⁺]_i and I_m were obtained under the conditions for electrophysiology recording as described previously. For synchronization of simultaneously acquired data, I_m data acquisition was triggered by the initiation of [Ca²⁺]_i data acquisition.

Immunocytochemistry

On d 3, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, treated with −20 C methanol, and blocked in 5% normal goat serum/PBS. Cells were exposed to 1:100 anti-SK2 and 1:500 anti-LH overnight at 4 C. Immunofluorescence staining with FITC- and TRITC-conjugated second antibodies was accomplished in successive incubations. As negative controls, either or both primary antibodies were omitted. To establish absence of overlap between the detection of FITC and TRITC, singly labeled cells were imaged under identical conditions as those for dual-labeled cells to confirm proper signal isolation. As specificity control, anti-SK2 was preincubated with SK2 peptide (1:1); when used under identical conditions, preabsorbed antibody showed no positive staining (data not shown).

LH secretion

On d 3, successive-timed incubations were collected before, during, and after three 15-min pulses of 1 nm GnRH. For the toxin-treated groups, either apamin or iberiotoxin was included in the medium beginning 15 min before the first GnRH pulse and continuing for 45 min. Samples were assayed for LH by RIA as described (14,19). The integrated secretory response to GnRH was calculated as the total amount of LH secreted during a 15-min exposure to GnRH plus that secreted in the subsequent 15 min.

Statistical analyses

Differences between two groups were analyzed with the t test using SigmaStat (Systat Software, San Jose, CA) or with the Welch modified two-sample t test using S-Plus (Insightful Corp., Seattle, WA). One-way ANOVA followed by Student-Newman-Keuls for multiple comparisons was used to examine toxin effects on LH secretion (SigmaStat). Two-way ANOVA was used to compare control and E₂ groups and the concentration-dependence of apamin inhibition of SK channel current (OriginPro; OriginLab Corp., Northampton, MA).

Results

Simultaneous determination [Ca²⁺]_i and I_m

In mouse gonadotrophs (20), as in other species (1), GnRH stimulates an increase in [Ca²⁺]_i. As shown in Fig. 1A, under voltage clamp the GnRH-induced increase in [Ca²⁺]_i precedes the increase in I_m; the [Ca²⁺]_i oscillations elicit synchronous changes in I_m. The I_m oscillations were of two general types and are demonstrated in the expanded time-scale insets in Fig. 1A. The distinguishing characteristics are in the kinetics of the decay to baseline of the I_m oscillations. In the first instance (Fig. 1A, left inset) the decay is relatively slow and follows the decline in [Ca²⁺]_i; in the second (Fig. 1A, right inset), the decay is biphasic with an initially fast component followed by a slower one. In all of the five voltage-clamped cells in which [Ca²⁺]_i and I_m were recorded simultaneously, one or more of the GnRH-induced I_m oscillations (eight of a total of 29 oscillations in the five cells) exhibited the initially rapid and then slower biphasic decay seen in Fig. 1A, right inset (also see Fig. 3A).

Figure 1.

Simultaneous determination of [Ca²⁺]_i and I_m in single mouse gonadotrophs. Representative [Ca²⁺]_i and I_m responses under voltage clamp (V_m = −50 mV) for control cells (no steroid pretreatment) to 1 nm GnRH (1-min exposure). A, Response to an initial GnRH pulse. The insets show the [Ca²⁺]_i and I_m relationships on an expanded time scale. The ordinates have been compressed to show temporal relationships; for both oscillations the increase in [Ca²⁺]_i precedes the increase in I_m. B, The same cell response to a second GnRH pulse (17-min interpulse interval) in the presence of 100 nm apamin. There is no I_m response to the initial [Ca²⁺]_i oscillation; however, the second [Ca²⁺]_i oscillation elicits a kinetically distinct fast, transient I_m response. The insets show the ordinate-adjusted oscillations on an expanded time scale; the increase in [Ca²⁺]_i precedes the increase in the apamin-insensitive I_m. s, sec.

Figure 3.

Iberiotoxin inhibition of apamin-insensitive I_m. For all records V_m was voltage clamped at −50 mV. Representative I_m records from individual gonadotrophs. A, Control response to GnRH (E₂ pretreatment) demonstrating the biphasic decay of I_m when the magnitude of peak I_BK exceeds that of I_SK. B, Response to GnRH (no steroid pretreatment) in the presence of 100 nm apamin reveals I_BK (upper panel). Following the same cell with GnRH in the presence of 100 nm apamin and 100 nm iberiotoxin shows I_BK inhibition (lower panel). C, Representative control records showing the I_BK response to GnRH (no steroid pretreatment) in the presence of 100 nm apamin (upper panel), followed by second response to GnRH in the presence of 100 nm apamin alone (no iberiotoxin, lower panel), showing that the absence of a response to GnRH in the presence of apamin plus iberiotoxin is due to I_BK inhibition and not simply a failure to respond to two successive GnRH pulses in the presence of apamin. The horizontal line below each trace indicates the time of 1 nm GnRH application. In B and C, the scale bars apply to both traces in the panel. s, sec.

We next determined the sensitivity of the GnRH-induced I_m to the toxin apamin, a specific blocker of Ca²⁺-activated SK channels (21,22,23). Figure 1B shows the response to a GnRH pulse in the presence of 100 nm apamin. In the five cells in which apamin was applied, the toxin did not alter the [Ca²⁺]_i response to GnRH but partially inhibited I_m in all five cells. Note that the residual, apamin-insensitive I_m has a distinct fast, transient kinetic profile as shown on an expanded time scale (Fig. 1B, right inset). Furthermore, the change in the shape of the decay of the second GnRH-induced I_m response in the presence of apamin (compare insets on right in Fig. 1, A and B) suggests that the more complex decay in the Fig. 1A inset (right) results from the presence of both apamin-sensitive SK channels and apamin-insensitive channel activity.

Apamin inhibition of GnRH-induced SK current (I_SK)

To further examine apamin inhibition of GnRH-induced current, we determined the concentration dependence of peak I_m inhibition by the SK channel-specific toxin. We have determined that the apamin-insensitive current shown in Fig. 1B is sensitive to the BK channel blocker iberiotoxin (see below); therefore, for this series the voltage-clamp potential was lowered to −66 mV to reduce the probability of activation of voltage- and Ca²⁺-sensitive BK channels. In addition, any I_m responses that exhibited the fast transient illustrated in Fig. 1 were omitted. Each cell served as its own control, receiving a control GnRH treatment followed by a second GnRH treatment in the presence of 0–100 nm apamin; intertreatment interval was no less than 15 min. Data for each cell were normalized (peak I_SK during second GnRH exposure/peak I_SK during first) to give the fraction of remaining current. Apamin inhibition of the normalized, fractional GnRH-induced I_SK was not different between control and E₂-pretreated cells (P = 0.64), indicating that the ability of apamin to inhibit I_SK is independent of E₂ pretreatment, and, therefore, data were pooled.

Figure 2A shows the concentration dependence of apamin inhibition. Half-maximal concentration for inhibition estimated from the fitted curve is 69 pm. Figure 2, B and C, shows representative records of concentration-dependent apamin inhibition of GnRH-induced I_SK for data with no contaminating BK current (I_BK). The control panels in Fig. 2, B and C, illustrate the variability in I_m response to GnRH between gonadotrophs as shown previously (2); however, for an individual gonadotroph, the GnRH response pattern is relatively consistent.

Figure 2.

Apamin inhibition of GnRH-induced peak I_SK and localization of SK2 to gonadotrophs. A, Concentration dependence of apamin inhibition of normalized, fractional I_SK under voltage clamp (V_m = −66 mV) and no contaminating I_BK (mean ± sem; n = 8–17; absence of an error bar indicates that the sem is smaller than the diameter of the symbol). For apamin-treated and control (zero apamin) cells, mean normalized, fractional remaining current data were fit to a logistic equation (weighted by the inverse of the variance) to obtain an estimate of the apamin concentration at half inhibition (IC₅₀). B and C, Representative voltage-clamp I_SK records from individual gonadotrophs showing the response to GnRH alone (upper panel), followed by GnRH in the presence of the indicated apamin concentration in the same cell (lower panel). The horizontal line below each trace indicates the time of 1 nm GnRH application. The scale bars apply to all traces. D, Colocalization of LH (red) and SK2 (green) to mouse anterior pituitary cells fixed and immunofluorescently stained with anti-LH and anti-SK2 (putative epitope location at intracellular C terminal) and analyzed by confocal laser scanning microscopy (Zeiss LSM 510, 63 × oil-immersion lens; Carl Zeiss MicroImaging, Inc., Thornwood, NY) with excitation as described (47). The same field is shown in the three panels. Far right, Overlay of both labels. Yellow is the result of colocalization of red and green labels, and demonstrates presence of SK2 in gonadotrophs. Note SK2 presence in other anterior pituitary cells. For each channel the pinhole was set to give an optical slice of 2 μm; field was optically sectioned with 20 overlapping slices to ensure acquisition of an image from the middle of each cell in the field. During the 4-d culture, pituitary cells retain rounded shape; therefore, depending on cell volume, images are captured at different depths for cells in this field. Representative of five separate experiments, 10 fields examined per experiment. s, sec.

In other systems, SK2 is the most sensitive SK isoform to the blocking action of apamin, reported in the 30- to 90-pm range, and next is SK3 at 500 pm or more (21,22,23,24). The results in Fig. 2 support the conclusion that SK2 is the predominant SK channel subtype responsible for the apamin-sensitive component of GnRH-induced I_m in mouse gonadotrophs; a significant presence of the other SK subtypes would shift the concentration dependence of inhibition to the right.

To examine for localization of SK2 channels to cultured gonadotrophs, dual-label immunofluorescent staining was used. As shown in Fig. 2D, cells that express LH, a gonadotroph marker, also express SK2. In five separate experiments, all cells staining positive for LH (n = 68) were positive for SK2. Also shown is SK2 presence in cells not containing LH, indicating that its expression in anterior pituitary cells is not gonadotroph exclusive.

Iberiotoxin inhibition of apamin-insensitive I_m

For these experiments, cells were voltage clamped at a more depolarized V_m, −50 mV, to increase the probability of activation of the voltage and Ca²⁺-sensitive BK channel in response to the GnRH-induced increase in [Ca²⁺]_i. Figure 3A shows the response to GnRH in an E₂-pretreated control cell (no toxin treatment) displaying biphasic oscillations with a decreasing fast component as the response proceeds from the first through the fifth oscillation. For toxin treatment each cell served as its own control, receiving an initial GnRH treatment in the presence of 100 nm apamin, followed by a second GnRH treatment in the presence of apamin only or apamin and 100 nm iberiotoxin, a selective, high-affinity inhibitor of BK channels (25). Figure 3, B and C, upper panels, shows I_m recordings from mouse gonadotrophs in response to GnRH (both cells, no steroid pretreatment) in the presence of apamin. The residual apamin-insensitive outward current is seen to activate and inactivate rapidly. As shown in Fig. 3B, lower panel, the apamin-insensitive outward I_m is inhibited when a second GnRH exposure includes apamin and iberiotoxin. The inhibition of the peak apamin-insensitive current at this iberiotoxin concentration was essentially complete [fractional current (second GnRH pulse/first): apamin + iberiotoxin/apamin, 0.10 ± 0.02, n = 7; apamin/apamin control, 0.81 ± 0.21, n = 7; P < 0.001; four E₂ and three no steroid-pretreated cells per group]. Figure 3C, lower panel, illustrates the apamin-insensitive I_m control response to a second GnRH pulse in the presence of apamin. These results support the conclusion that the residual apamin-insensitive outward I_m in mouse gonadotrophs is I_BK.

Figure 4 compares the kinetic behavior of the mouse gonadotroph currents identified as I_BK and I_SK. When individual oscillations for these two currents are normalized to peak current height and to the time when they reach one half of the normalized peak, it is evident that mouse gonadotroph apamin-sensitive I_SK and iberiotoxin-sensitive I_BK have distinct, easily recognized kinetic profiles (Fig. 4A). Quantitative analysis of two characteristics is presented in Fig. 4B. In the absence of toxin(s), the coexistence of I_SK and I_BK in I_m records is not always evident, at least in part, because I_BK is transient and can be obscured by the longer duration I_SK. However, when V_m was voltage clamped at −50 mV to increase the probability of I_BK activation and 100 nm apamin was present, I_BK was found in 65% (17/26) of no-steroid pretreated cells and 67% (14/21) of E₂-pretreated cells selected as presumptive gonadotrophs that subsequently responded to a GnRH challenge. This is similar to our overall success rate in this study (62%) for selecting mouse gonadotrophs from mixed cell populations of dispersed mouse anterior pituitary cells. These data indicate that I_BK is consistently expressed and readily separated from I_SK in mouse gonadotrophs.

Figure 4.

Kinetics of I_SK and I_BK. A, Single I_SK and I_BK oscillations (SK inset from Fig. 2B, upper panel, and BK inset from Fig. 3C, upper panel) on an expanded time scale and normalized to maximum peak height and the time at which one half of the peak height was attained. B, Quantitative analysis of kinetic variables for I_BK and I_SK oscillations shows significant differences in the time required to increase to peak value (*, P = 0.013; n = 10 and 7) (left) and the width in ms (msec) of the oscillation when it is has increased to one half of the peak value (**, P < 0.001; n = 10 and 7) (right). A single I_m oscillation was analyzed for each cell. Records that had been baseline corrected and filtered at 100 Hz post-acquisition were analyzed for the rise time to peak as an estimate of the speed of activation and for half-width (oscillation width in ms at one half of the peak height) as an estimate of oscillation duration. s, sec.

E₂ modulation of I_SK and I_BK

Our previous study showing that E₂ pretreatment reduces the GnRH-induced peak outward I_m in voltage-clamped mouse gonadotrophs (2) was expanded to analyze effects specific to the two components, I_SK and I_BK. For I_SK we analyzed records obtained at V_m = −66 mV, in which the transient I_BK component was not present; E₂ pretreatment significantly attenuates GnRH-induced peak I_SK (Fig. 5), as well as decreasing the mean current during the response and the current area per oscillation (supplemental Table 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo. endojournals.org). In marked contrast, analysis of peak I_BK in voltage-clamped (−50 mV) mouse gonadotrophs in the presence of apamin to inhibit I_SK showed that E₂ pretreatment significantly augments peak I_BK in mouse gonadotrophs (Fig. 5).

Figure 5.

E₂ effect on I_SK and I_BK. SK: control (no steroid) and E₂ (0.2 nm) pretreated gonadotrophs voltage clamped at −66 mV were challenged with 1 nm GnRH. Peak I_SK was significantly reduced by E₂ pretreatment (*, P < 0.008; control, n = 41; E₂, n = 34). The C_m (membrane capacítance), baseline I_m, the number of oscillations, and their frequency were not affected by E₂ pretreatment (supplemental Table 1). BK: control (no steroid) and E₂-pretreated (0.2 nm) gonadotrophs voltage clamped at −50 mV were challenged with 1 nm GnRH in the presence of 100 nm apamin to inhibit I_SK. Peak I_BK was significantly augmented by E₂ pretreatment (**, P < 0.04; control, n = 16; E₂, n = 12).

SK and BK channels and LH secretion

We next asked whether pharmacological blockade of SK or BK channels, and a presumptive change in membrane excitability, would affect the ability of GnRH to stimulate LH secretion. In addition, because of the differential action of E₂ on peak I_SK and I_BK induced by GnRH (Fig. 5), the LH secretory responses were determined for cells cultured with or without E₂ pretreatment. The context for this series is the demonstrated ability of preovulatory E₂ levels to increase the gonadotroph secretory response to GnRH in vivo and in vitro (e.g. Refs. 14 and 26,27,28). Consistent with E₂-positive action on LH secretion, mouse pituitary cells cultured in a background of 0.2 nm E₂ respond to three hourly pulses of GnRH with LH secretion that is significantly greater (P < 0.001) than that found in the absence of added steroid (Fig. 6B).

Figure 6.

LH secretion. A, Effect of SK or BK channel blockade on GnRH-stimulated LH secretion. After 3 d culture without (upper graph) or with (lower graph) 0.2 nm E₂, LH secretion from mouse anterior cells was monitored in response to 15-min pulses of 1 nm GnRH. For the toxin-treated groups, either 100 nm apamin or 100 nm iberiotoxin was included in the medium beginning at 15 min before the first GnRH pulse and continuing a total of 45 min. Results are expressed as mean ± sem from three to six independent experiments per treatment group. Where not shown, the error bar is smaller than the symbol representing the mean. B, Effect of E₂ (0.2 nm, 3 d) on LH secretion in response to three 15-min pulses of 1 nm GnRH with 1-h interpulse intervals. Secretion rates for pulses 1 and 2 are shown in A [no-toxin ctrl (control)]. Mean ± sem from six independent experiments per treatment group. *, Significantly different from no steroid (P < 0.001).

Addition of the SK blocker apamin to pituitary cultures 15 min before the first GnRH pulse resulted in a significantly enhanced LH secretory response that was 2-fold for no-steroid treated cells (Fig. 6A, upper graph; P < 0.001) and 3-fold for cells with an E₂ background (lower graph; P < 0.001) compared with no-toxin control. Regardless of the steroid background, apamin addition had no significant effect on baseline secretion, and the effect on GnRH-stimulated secretion was reversible as evidenced by the temporal profile of stimulated-LH secretion and the response to a subsequent GnRH pulse, which was indistinguishable from control.

Addition of iberiotoxin to block BK channel activity had no significant effect on GnRH-stimulated LH secretion for cells cultured without added steroids (Fig. 6A, upper graph). However, for cells pretreated with E₂, BK channel blockade with iberiotoxin resulted in a significant reduction in GnRH-stimulated LH secretion (P = 0.011) (Fig. 6A, bottom graph).

Discussion

We previously reported that GnRH induces a large amplitude outward I_m in voltage-clamped mouse gonadotrophs that is characteristic of current flow through Ca²⁺-activated K⁺ channels (2). In addition, we found that the magnitude of this current is suppressed by E₂ pretreatment, suggesting that a decrease in Ca²⁺-activated K⁺ channel activity to increase membrane excitability is one mode of action of E₂ in modulating gonadotroph function. An objective of the present study was to identify the specific Ca²⁺-activated K⁺ channel types in mouse gonadotrophs that could link the GnRH-induced increase in [Ca²⁺]_i to changes in plasma membrane electrical activity. Using simultaneous measurement of [Ca²⁺]_i and I_m in voltage-clamped mouse gonadotrophs, we show that GnRH stimulates an increase in [Ca²⁺]_i that precedes the outward I_m. For this outward I_m, we found that the GnRH-stimulated increase in [Ca²⁺]_i activates two kinetically distinct outward currents that were identified as I_SK and I_BK.

Ca²⁺-activated K⁺ channel identification

Single-channel conductances distinguish members of the Ca²⁺-activated K⁺ channel family, and subfamily members are further differentiated by distinct kinetic and pharmacological properties. Work defining SK members largely has been possible because of the toxin apamin, for which the only known receptors are SK channels (23). The three SK channel subtypes have different sensitivity to the blocking action of apamin, with SK2 > SK3 ≫ SK1. We show that the apamin-sensitive current in mouse gonadotrophs has an IC₅₀ consistent with SK2 channel data in other systems (21,22,23,24), and SK2 presence is confirmed by immunocytochemistry. The unique toxin sensitivities of BK as well as SK2 channels allowed us to establish that the two are jointly expressed in mouse gonadotrophs and are components of GnRH-induced outward I_m.

I_SK and I_BK kinetics

A striking observation in this study is the difference in the kinetic profiles of the current oscillations for I_BK (fast transient) and I_SK (longer duration). The basis for the marked differences in I_SK and I_BK kinetic behavior is an intriguing unknown, but BK and SK channel characteristics suggest some interesting possibilities. Because these experiments were done under voltage clamp, voltage-activated events are eliminated, voltage-gated Ca²⁺ entry across the plasma membrane does not occur, and the activation of voltage- and Ca²⁺-sensitive BK channels is limited to Ca²⁺ at a fixed voltage. Under these conditions SK and BK channels are responding only to the GnRH-stimulated release of Ca²⁺ from intracellular stores. This mimics gonadotroph function in that secretion is initiated by release of Ca²⁺ from intracellular stores, and is independent of and relatively insensitive to EC Ca²⁺ entry through voltage-gated Ca²⁺ channels (1). SK channels are sensitive to [Ca²⁺] in the submicromolar range consistent with the observation that I_SK tracks global [Ca²⁺]_i in the rat (3) and with the I_SK profiles in the mouse as shown here. For I_BK, we show that the transient current is too fast to reflect fully the time course of the change in global [Ca²⁺]_i. There are at least two, not necessarily exclusive, explanations. First, BK channel activation generally requires higher [Ca²⁺] than that for SK channels. If BK channels and intracellular Ca²⁺ release sites are colocalized, the rapid closure of BK channels may reflect the rise and fall of [Ca²⁺] in the microdomain near the channels (29). Although microdomain relationships are not known for BK channels, SK channels and intracellular Ca²⁺ release sites colocalize in the rat gonadotroph (1). Second, BK channels may close despite the continued presence of elevated [Ca²⁺]_i. Regarding this possibility, inactivation of BK channels can occur as a result of coexpression of the pore-forming α-subunits with either the β2 or a β3 splice variant (13,30). Expression of specific BK-β subunits has not been established for gonadotrophs.

SK channel function

A general function of SK and BK channels is integration of changes in [Ca²⁺]_i with changes in membrane potential. The specific functional consequences of this interplay depend on cell type and association with other channels. The consequence of SK channel activation generally is V_m hyperpolarization and a decrease in membrane excitability. For example, SK channels in myometrial cells are involved in regulating uterine contractility (31), in vascular endothelial cells are reported to regulate agonist-induced nitric oxide synthesis (32), and in neurons have a role in regulating firing frequency (11). For endocrine cells a decrease in SK channel activity and, thus, increased electrical excitability would be expected to increase agonist-stimulated secretion. As we report here for gonadotrophs, one outcome of the presumptive increase in excitability when SK channel activity is reduced by apamin is marked enhancement in GnRH-stimulated LH secretion. Similar outcomes have been reported for adrenal chromaffin cells; apamin inhibition of SK channels augments catecholamine secretion stimulated by muscarinic or nicotinic agonists or pituitary adenylate cyclase-activating polypeptide (33,34,35). To our knowledge the present study is the first to demonstrate the consequences of SK channel interference for LH secretion. However, studies with rat lactotrophs and the GH3 rat cell line report that apamin blockade can further increase the elevated secretion of prolactin found in the absence of dopamine but does not augment secretion induced by TRH (36,37).

BK channel function

The contribution of BK channel activation to membrane excitability is more complex, depending on cell type and expression of auxiliary β-subunits. The overall function of BK channels is the integration of [Ca²⁺]_i signals with changes in membrane potential leading to negative feedback for limiting depolarization-driven Ca²⁺ influx. However, differential expression of β-subunits can confer striking differences in gating and kinetics of the pore-forming α-subunits, contributing to a variety of physiological roles. For example, in neurons BK channels with auxiliary β4-subunits are thought to down-regulate neurotransmitter release by shortening the duration of depolarization associated with Ca²⁺ entry (38); in vascular smooth muscle cells, which predominantly express the β1-subunit, I_BK-induced extended hyperpolarization leads to relaxation critical for control of vascular tone (39). In contrast, BK channels in cells that predominantly express the β2 auxiliary subunit exhibit rapid inactivation, allowing for repetitive firing with a presumptive positive consequence to secretion in, for example, adrenal chromaffin cells (40).

For somatotrophs, the role of BK channels in shaping the action potential waveform has been examined (1), but there is no information on channel kinetics implicating the association with a specific β-subunit. As shown here for mouse gonadotrophs, BK channels exhibit rapid inactivation, and the kinetic profile is consistent with that found when β2 is the predominate subunit in other cell types. Also in line with that possibility, BK channel blockade decreased GnRH-stimulated LH secretion but notably only when the gonadotrophs had been treated with E₂.

E₂ modulation of SK and BK channels

Additional regulators of SK and BK channel function are cell-specific factors modulating channel expression or activity. Previously, we showed for mouse gonadotrophs that E₂ attenuates GnRH-induced, here identified as I_SK, as well as voltage-activated I_m (2). Conversely, for BK channels, E₂ pretreatment augments peak I_BK. A potential physiological consequence of this differential action of E₂ is evident in the LH secretory response after selective removal of either SK or BK. The augmentation of GnRH-stimulated LH secretion occurring when SK channels are blocked can be interpreted as reflecting increased membrane excitability (decreased I_SK) and prominence of I_BK with its rapid inactivation that can promote abbreviated hyperpolarization; in E₂-treated cells under the same SK channel blockade, the even greater augmentation of LH secretion could be a reflection of the E₂-induced increase in functional BK channel density. Consistent with this interpretation, when the compensating effect of BK channel kinetics is pharmacologically blocked in E₂-treated cells, the consequence is a decrease in stimulated LH secretion.

Not addressed are possible mechanisms for E₂ modulation of SK and BK channels. In other systems the focus primarily has been on regulation of SK or BK expression (e.g. Refs. 31 and 41,42,43). However, for SK channels, evidence suggests that SK2 binding components are targets for modulation. SK2-associated calmodulin can be phosphorylated by another member of the multiprotein channel assembly, casein kinase 2 (CDK2), resulting in a change in channel activity (44). The challenge is identification of potential modifiers of CDK2 and protein phosphatase 2A, another assembly component. No studies have tested the possibility that E₂ can affect SK2 channel activity through modulation of SK2-associated CDK2 activity; however, there is suggestive evidence for estrogen receptor α/ERK/MAPK-mediated increase in CDK2 activity in endocervical cells (45). For BK channels a subnanomolar E₂ background as used here has been shown to selectively increase expression of α- and β4-subunits in a GnRH-secreting neuronal cell line, GT1–7 (43). In addition, a number of splice variants of BK-α and the β-subunits have been identified to be under hormonal regulation and associated with distinct channel properties (12); e.g. an alternatively spliced BK-α, which reverses phosphorylation regulation, is expressed in pregnant myometrium under E₂ control (46). Mechanisms underlying the action of E₂ on I_SK and I_BK in gonadotrophs remain to be determined.

In summary, we find that the GnRH-induced, Ca²⁺-activated outward I_m in mouse gonadotrophs is carried by both BK and SK Ca²⁺-activated K⁺ channels. Furthermore, these currents are differentially modulated by E₂ pretreatment. Inhibition of SK or BK channels affects GnRH-stimulated LH secretion, implicating more directly a physiological role for these channels in gonadotroph function, including as targets for E₂ positive feedback on LH secretion. The effect on membrane electrical excitability in gonadotrophs of a GnRH-stimulated increase in [Ca²⁺]_i includes the interplay of I_SK and I_BK activation. Contributions of these currents during GnRH-induced [Ca²⁺]_i oscillations will reflect their individual expression level and modulation by E₂, their activation timing as reflecting their [Ca²⁺] sensitivity, their inactivation kinetics, and their spatial relationship to Ca²⁺ release sites.