Abstract
BackgroundGranulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca2+-activated K+ channel (KCa) of big conductance (BKCa), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of KCa channels (including BKCa beta-subunits), and 2. biophysical properties of BKCa channels.MethodsGCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique.ResultsWe identified two KCa types in human GCs, the intermediate- (IK) and the small-conductance KCa (SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by KCa blockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single KCa channels with distinctive features. Both, IK and BKCa channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BKCa channel revealed the presence of mRNAs encoding several BKCa beta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BKCa channels which we observed in electrophysiological recordings.ConclusionFunctional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BKCa, they express all three KCa types known. We suggest that the plurality and co-expression of different KCa channels and BKCa beta-subunits might allow differentiated responses to Ca2+ signals over a wide range caused by various intraovarian signalling molecules (e.g. acetylcholine, ATP, dopamine). The knowledge of ovarian KCa channel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.
Highlights
Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones
As big conductance K+ channel (KCa) (BKCa) channel characteristics are affected by the type(s) of accessory β subunits present, we studied their expression in human GCs as well
Role of KCa channels in steroidogenesis in human GCs Functionality of BKCa channels is known to be necessary for steroidogenesis in human GCs [8]
Summary
Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. We identified in human GCs a Ca2+-activated K+ channel (KCa) of big conductance (BKCa), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca2+ levels. Acetylcholine (ACh) is produced by and 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]. 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. Activation of Ca2+activated ion channels such as the BKCa is a well-known consequence of raised [Ca2+]i [13,14,15,16,17]
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