Reproductive Biology and Endocrinology Open Access Identification and Characterization of Ca2+-activated K+ Channels in Granulosa Cells of the Human Ovary

Background: Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca 2+-activated K + channel (K Ca) of big conductance (BK Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca 2+ levels. In this study, we aimed at characterizing 1. expression and functions of K Ca channels (including BK Ca beta-subunits), and 2. biophysical properties of BK Ca channels.


Background
Ion channels of ovarian granulosa cells (GCs) have been identified and functionally characterized in only a few species (human, swine, chicken) ( [1][2][3] and references in [4]). In human and porcine GCs, several channel types are involved in the physiologically important process of progesterone production [4][5][6][7]. A K Ca channel of large (big) conductance (BK Ca ) in human GCs has a part in endocrine-regulated progesterone production [8]. Blocking of BK Ca channels results in reduction of human chorionic gonadotropin (hCG)-induced progesterone production, but does not affect basal steroidogenesis.
Moreover, BK Ca channels in human GCs were shown to be opened by cholinergic and oxytocinergic stimulation entailing transient membrane hyperpolarisation [8]. Acetylcholine (ACh) is produced by and also acts upon human GCs. In the human ovary, the non-neuronal cholinergic system affects several physiological functions, e.g. cell proliferation, gene transcription, and intercellular communication via gap junctions [9]. It represents just one of the local signaling systems which have been identified in recent years to complement endocrine (FSH, LH/hCG) and neuronal control of ovarian functions via autocrine/paracrine pathways. Besides ACh, other local intraovarian signaling molecules are peptide hormones (e.g. oxytocin, relaxin), catecholamines (e.g. norepinephrine, dopamine), ATP, prostaglandins, GABA and histamine. Many of these compounds (e.g. ACh, oxytocin, relaxin) exert their actions upon GCs via alteration of intracellular Ca 2+ levels [Ca 2+ ] i [8][9][10][11][12]. In human GCs, ACh and its agonist carbachol activate muscarinic receptors (e.g. M1) and increase [Ca 2+ ] i via Ca 2+ release from intracellular stores [9]. Activation of Ca 2+activated ion channels such as the BK Ca is a well-known consequence of raised [Ca 2+ ] i [13][14][15][16][17].
Up to now, three K Ca families are known, which are classified by their single channel conductance: SK (small conductance K Ca ), IK (intermediate conductance K Ca ) and BK Ca [13,[15][16][17][18]. They differ also regarding molecular and biophysical properties as well as their regulation by pharmacological compounds. For each class specific blockers exist which do not affect the two other classes. Three different apamin-sensitive SK channels were cloned (SK1, SK2, SK3), which exhibit single-channel conductances of g sc = 2-20 pS [18]. The IK with g sc = 10-80 pS is sensitive to TRAM-34 and was found in only a few non-neuronal cells, e.g. epithelial cells and erythrocytes ('Gárdos channel') [16,[19][20][21]. The BK Ca channel has one of the highest known single channel conductances of g sc = ca. 200 pS and is sensitive to iberiotoxin (IbTx). It is the only subtype of the K Ca family that exhibits a pronounced voltage-dependence in addition to its Ca 2+ -sensitivity [13,14,16,[22][23][24].
Our study aimed at the investigation of K Ca channels in human GCs since one member of this channel group, i.e. the BK Ca channel, has a part in both endocrine (i.e. role in hCG-stimulated steroidogenesis) and autocrine/paracrine pathways (i.e. cholinergic and oxytocinergic activation). The human GCs investigated originate from pre-ovulatory human follicles obtained from patients undergoing in vitro-fertilization (IVF). They represent a cell culture model of their in vivo counterparts in the antral follicle and the young active corpus luteum (CL). The study shall provide data on molecular characterization of other members of the K Ca channel family (SK, IK) in human GCs and their potential role in steroidogenesis. Furthermore, the Ca 2+ -and voltage-dependence of the BK Ca channel was determined in electrophysiological single-channel recordings. As BK Ca channel characteristics are affected by the type(s) of accessory β subunits present, we studied their expression in human GCs as well. There are four potential types known (β1, β2, β3, and β4), which interact with the α subunit and regulate BK Ca channel function regarding impact of Ca 2+ and voltage [25][26][27][28][29]. They represent also binding sites for toxins and drugs as well as phosphorylation sites [26,27,[30][31][32].

Human GC preparation and culture
Human GCs were isolated from follicular aspirates of women undergoing IVF and cultured in DMEM/F12 (10% FCS; Sigma-Aldrich, Munich, Germany) under a humidified atmosphere at 37°C/5% CO 2 [33]. Use of cells was approved by the patients and the Ethics Committee of the University of Munich. All patients were treated following standard IVF protocols and negatively diagnosed for the polycystic ovary syndrome. To account for patient-topatient variations all studies were performed on several, randomly selected cell preparations (pooled from up to 3 patients) on different days.

Human tissue samples
Human ovarian samples containing CL from consenting patients undergoing gynecological surgery (generously provided by C. Heiss, Klinik am Eichert, Göppingen, Germany) were fixed in Bouin's fixative and embedded in paraffin. Apart from this, we used paraffin-embedded ovarian samples with follicles from the tissue archive of the Women's Hospital in Munich, which had been taken from pre-menopausal women during autopsies [34]. The Ethics Committee of the University of Munich approved all procedures concerning use of human materials. Pathological deterioration of follicles and corpora lutea in these human ovarian samples were excluded by morphological and microscopical assessment.

Progesterone assay
Progesterone concentrations were measured in supernatants of human GCs cultured in 24-well plates. On day three of culture, cells were treated in triplicates with the respective compounds. After 24 h the supernatants were collected and the progesterone concentrations were measured using an ELISA (DRG Instruments, Marburg, Germany). Experiments were repeated with cells from independent cell preparations (each pooled from up to 3 patients) to account for interpatient variability. After normalization to the respective control (untreated) values, data were statistically analyzed by a repeated measures ANOVA followed by Newman-Keuls multiple comparison test.

Electrophysiology
Human GCs were grown on glass cover slips for 2-12 days and currents were recorded at room temperature by means of an EPC-9 amplifier (HEKA elektronik, Lambrecht, Germany; sample rate, 10 kHz; low pass filter, 2.5 kHz) [8]. Positive currents represent outward currents and all potentials given refer to the cytoplasmic side of the plasma membrane. Potentials were corrected for a liquid junction potential of +16 mV [35]. The extracellular solution (EC) contained (in mM) 140 NaCl, 3 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 Hepes, and 10 glucose (pH 7.4). The intracellular solution (IC) contained (in mM) 130 K-gluconate, 5 NaCl, 2 EGTA, 1 MgCl 2 , and 10 Hepes (pH 7.4). Single channel currents were recorded in the inside-out configuration. To assess the Ca 2+ -sensitivity of the BK Ca channel the free Ca 2+ concentration [Ca 2+ ] i of the IC solutions was adjusted by using varying concentrations of CaCl 2 according to calculations performed by means of the software "Calcium" [36]. To obtain channel current-voltage relationships a voltage protocol ranging from -80 mV to +90 mV in 10 mV steps was used with each potential applied for 200 ms. For evaluation of channel open probability P o , single channel currents were recorded at +60 mV, +70 mV, +80 mV, and +90 mV for 1600 ms in each case. For analysis, frequency histograms of current traces were calculated and the two amplitude peaks corresponding to open and closed channel state were fitted using Gaussian distributions. The single channel current amplitude was measured as differences between the two peaks. Integration of the area under the curves and division of the area under the open channel peak by the area under the entire curve yielded P o . In the case of two or more active individual channels in an inside-out recording, the open probability per one chan-nel was calculated by converting the values according to the number of simultaneously active channels at each time point. [Ca 2+ ] i values for half-maximal activation (EC 50 ) were determined by fitting P o as a function of [Ca 2+ ] i with a sigmoidal dose-response-curve with variable Hill slope and a fixed bottom value of zero. The voltage-dependence of P o was fitted using a Boltzmann sigmoidal function with the bottom value fixed to be 0 and potentials for half-maximal activation (V 50 ) as well as slope values were obtained. The values are given with the respective 95% confidence interval (C.I.).

RT-PCR
Total RNA was isolated from several human GC preparations, and reverse transcribed using Superscript-RT II (Life Technologies, Karlsruhe, Germany) in combination with either a 18-mer polydeoxythymidine primer or random hexamers of polydeoxynucleotide primers. PCR amplification was carried out with oligodeoxynucleotide primer pairs, which spanned at least one intron of the genomic sequence (except for the second BK β1 primer pair; Table  1) [37,38]. The PCR protocol consisted of 35 cycles of denaturation at 94°C (30 s), annealing at the temperatures given in Table 1 (30 s), and elongation at 72°C (45 s) using a PTC-200 Peltier Thermal Cycler (MJ Research, Bio-Rad, Munich, Germany). PCR products were separated on an agarose gel and visualized by ethidium bromide staining and ultraviolet illumination. Identity of all PCR products was verified by sequencing (Agowa, Berlin, Germany).

cDNA array
Total RNA of GCs cultivated for 3 days was isolated and subjected to the GEArray Q Series Human Neuroscience-1 Ion Channel and Transporter Gene Array (SuperArray Bioscience Corp., Frederick, MD). The cDNA array was analyzed by means of a chemiluminescent detection method (Roche Diagnostics, Mannheim, Germany) [39].

Cytotoxicity assays
Potential cytotoxicity of the channel blockers was evaluated by using commercial non-radioactive cell proliferation assays (CellTiter and CellTiter-Glo, both from Promega, Mannheim, Germany) [11]. Human GCs were cultured in 24-or 48-well plates and treated in triplicates for 24 h on day 3 of culture with the substances applied for progesterone measurements.

Data analysis
Data were analyzed and depicted using Prism 4 (Graph-Pad Software, San Diego, California) and SigmaPlot 10 (SPSS, Chicago, Illinois). Data represent means ± SEM if not stated otherwise.

Role of K Ca channels in steroidogenesis in human GCs
Functionality of BK Ca channels is known to be necessary for steroidogenesis in human GCs [8]. Blocking of IK channels by TRAM-34 (1 μM) and of SK channels by apamin (100 nM) had an inhibitory action on hCG (10 IU/ml)-induced progesterone production as well ( Figure  1). The blockers had no effect on basal progesterone production. This casts cytotoxic actions of the blockers into doubt. In addition, neither two different proliferation/ cytotoxicity assays nor inspection of the cells by light and electron microscopy showed any detrimental alterations caused by the K Ca blockers (data not shown).

Expression of different K Ca channel families in human GCs
The presence of mRNAs coding for all known types of SK channels and the IK channel was demonstrated by cDNA arrays (except for SK2) and by RT-PCR followed by sequencing (Figure 2A). In case of SK1 two PCR products were found and sequenced, which both correspond to the respective channel. They differ with regard to presence or absence of exon 9 (the primers match sequences in exon 8 and 10, respectively) and are, thus, most likely yet unknown splicing variants. IK and SK2 α subunit proteins are expressed in GCs of the human ovary ( Figure 2). Expression of SK1 and SK3 α subunits cannot be unambiguously appraised since the antisera used for immunohistochemistry produced unspecific (e.g. nuclear) staining (data not shown).
In electrophysiological recordings in the inside-out configuration, a second type of K Ca channel besides the BK Ca was recorded at [Ca 2+ ] i > 1 μM (Figure 3). This channel is most likely the IK channel because of its characteristic intermediate single-channel conductance of 67 ± 5 pS and a reversal potential of V 0 = 7 mV under symmetrical K + -gluconate concentrations (n = 5; Figure 3B), its increasing P o with rising [Ca 2+ ] i (data not shown), and because K + was the only permeant ion present in sufficiently high concentrations on both sides of the membrane. The max-iCl can be excluded due to the low symmetrical Cl --concentrations of 9 mM. In almost all recordings exhibiting the IK channel, at least one BK Ca channel was simultaneously active ( Figure 3A).  potentials (+70 mV) inactivation of BK Ca channels was observed for time periods ranging from several hundred ms to several s ( Figure 4C). Periods of inactivation longer than 500 ms were not considered when calculating P o ; only the trace before onset of inactivation was evaluated.

Ca 2+ -and voltage-dependence of BK Ca channels in human GCs
The voltage-dependence of the BK Ca channel was determined at high [Ca 2+ ] i because at lower [Ca 2+ ] i the channel was not active at negative potentials. By fitting the data with a Boltzmann sigmoidal function a potential for halfmaximal activation of V 50 = -54 mV (-56 to -51 mV, 95%-C.I.) and a slope factor of +12 mV (+9 to +15 mV) were observed at [Ca 2+ ] i = 1 mM (n = 4; Figure 4D).

Expression of BK Ca β subunits in human GCs
By RT-PCR and subsequent sequencing, we identified mRNAs encoding the BK Ca subunits β2, β3, and β4 in human GCs ( Figure 5A). The β1 subunit was not detected using two different primer pairs, which amplified the β1 subunit in positive control tissues (ovary, prostate, testis, colon, heart, and lung; data not shown). The BK Ca β4 subunit protein was also found in endocrine cells of the human CL by means of immunohistochemistry ( Figure 5B).

Discussion
In the present communication we report that human GCs possess in addition to the BK Ca other functional K Ca channels. The detection of mRNAs encoding the intermediateconductance K Ca (IK) as well as all three known types of small-conductance K Ca (SK1, SK2, SK3) points at a complex K Ca repertoire. The finding of mRNA alone can be misleading as was shown in a study on glioma cells in which mRNAs for Various K Ca channels are expressed in human GCs    all K Ca channels were detected, although only BK Ca channels were functional [41]. However, in human GCs all three classes of K Ca channels are functional, because specific blockers attenuated hCG-stimulated steroidogenesis. The IK blocker TRAM-34 was recently reported to inhibit nonselective cation channels as well [42], but is still one of the most accepted pharmacological tools to block IK channels. We cannot definitely exclude a role of nonselective cation channels. However, as they were not found in human GCs so far and because we presented further molecular proof of IK presence, we interpret the TRAM-34 action as blockage of IK channels. In addition, single K Ca channels were recorded which we identified as IK based on Ca 2+ -dependence and single channel conductance. A role of IK and SK channels in vivo is assumed because the corresponding proteins (IK, SK2) were detected -like BK Ca [8] -in endocrine cells of the human ovary. Despite mRNAs coding for all three known SK channels were found, we can only conclude that at least one of them is functional due to the impact of the SK blocker apamin on hCG-induced steroidogenesis. As the SK2 protein was detected in human ovarian slices it is likely that this type (and probably others) is present in cultured human GCs.
Besides their role in endocrine stimulated steroidogenesis, K Ca channels in human GCs represent a link to local regulatory systems of the ovary operating via signaling molecules (ACh, oxytocin, relaxin, norepinephrine, dopamine, ATP) that are known to alter [Ca 2+ ] i [10][11][12]. Therefore, and because the BK Ca is the best studied K Ca channel, we assessed its Ca 2+ -and voltage-dependence by means of single-channel recordings. The BK Ca channel was half-activated at about 20 μM [Ca 2+ ] i at +60 mV. An increase of [Ca 2+ ] i by one order of magnitude is known to shift the potential V 50 necessary for half-maximal activation in several other cell types by about 50 mV (30 to 94 mV) to more negative values [43][44][45]. At a global [Ca 2+ ] i = 1 μM that can be reached by stimulation with ACh, oxytocin, or relaxin [8][9][10], the potential V 50 necessary for half-maximal activation can be estimated to equal +100 mV. Although this represents a rather non-physiological voltage value, BK Ca channels can be activated under physiological conditions in human GCs since its specific blocking with IbTx attenuates hCG-stimulated progesterone production [8].  [46,47].
The variability of EC 50 values for Ca 2+ -sensitivity of the BK Ca channel over two orders of magnitude could be due to different β subunits present since they are known to be important modulators of Ca 2+ -and voltage-sensitivity of the channel [25][26][27][28][29][30][31][32]48]. Similar variations of EC50 and half-maximal activation voltages were reported for BK Ca in other cell types [13,15,43,45,[49][50][51]. Therefore, we have studied the BK Ca β subunit expression in human GCs and found mRNAs encoding the accessory subunits β2, β3, and β4, but not β1. Reports on the presence of β2, β3 and β4 mRNAs in material from whole human ovaries can now be better interpreted in terms of a GCs contribution to the mRNA findings [29,31].
The consequences of BK Ca β subunit expression pattern for channel function in human GCs are difficult to compare with studies in cellular model systems expressing only one type of β subunit. Nevertheless, the β subunit repertoire found on the mRNA level might help to explain our observations. The recorded inactivation of BK Ca channels at high positive potentials and at high free [Ca 2+ ] i was reported to occur in the presence of β2 and/or β3 subunits [26][27][28]52,53]. The absence of the β1 subunit might explain why oestrogens do not activate the BK Ca in human GCs [8]; in contrast to myometrial smooth muscle in which activation by oestrogens was ascribed to the presence of β1 [54,55]. The presence of β4 should introduce IbTx-resistance to BK Ca channels [32]. However, the fact that IbTx blocks both BK Ca whole-cell currents and hCG-Expression of BK Ca β subunits Figure 5 Expression of BK Ca β subunits. (A) In human GCs, mRNAs coding for several β subunits were detected. In negative controls (Co) template was replaced by water. (B) BK Ca β4 subunit protein is present in GCs of human CL. Bar, 50 μm. stimulated progesterone production points at a more complex picture, i.e. at least a proportion of the BK Ca channels in human GCs is IbTx-sensitive and, thus, probably contains not only β4 [8].
The simultaneous presence of different K Ca types is known from other cell types [18]. But what could be the cellular relevance of the ostensible redundancy to have different K Ca channels? They differ regarding regulation, biophysical properties as well as Ca 2+ sensitivity with IK and SK channels being activated at lower [Ca 2+ ] i than the BK Ca [17,18]. In addition, the BK Ca is voltage-sensitive in contrast to other K Ca channels [13,14,16,24], which would allow differentiated responses to the same Ca 2+ signals at varying membrane potentials. Concerning the multitude of K Ca channels in human GCs the question arises whether each individual GC expresses all identified K Ca channel subunits in parallel. The RT-PCR and progesterone production experiments can provide no answers about single cells. But single channel recordings revealed that at least BK Ca and IK channels can be present in the plasma membrane of the same individual cells. However, it is very likely that individual GCs can exhibit a varying K Ca repertoire and that GC subpopulations might exist regarding the expression of β subunits. Immunohistochemical results are in favor of such an assumption, since for IK, SK2, and β4, the degree of immunostaining in GCs of the human CL varies. The variations in Ca 2+ -sensitivity observed in single BK Ca channel recordings might also reflect BK Ca heterogeneity in single GCs and/or between individual GCs.

Conclusion
In summary, we found expression (in vitro, ex vivo) of several classes of K Ca channels in human GCs, which are all involved in gonadotropin-stimulated sex steroid hormone production. The presence of different K Ca channels and the observed heterogeneity in Ca 2+ -sensitivity of the BK Ca channel, which is probably due to expression of various β subunits, could allow finely tuned and differentiated cellular responses over a wide [Ca 2+ ] i range. The question of existence of GCs subpopulations regarding K Ca channels and BK Ca β subunits has to be studied in the future to understand cellular processes on the level of individual GCs. The rich instrumentation of Ca 2+ -dependent channels might be seen in relation to the abundance of intraovarian signaling molecules (e.g. ACh, ATP, dopamine, oxytocin, relaxin) acting via raised Ca 2+ levels. Therefore, we suggest that this channel group has a part in mediating the conjunction between endocrine (hCG, LH) and local ovarian signaling systems.