Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

Granulosa 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 (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 Ca²⁺ 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.

GCs 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.

We identified two K_Ca types in human GCs, the intermediate- (IK) and the small-conductance K_Ca (SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by K_Ca blockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single K_Ca channels with distinctive features. Both, IK and BK_Ca 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 BK_Ca channel revealed the presence of mRNAs encoding several BK_Ca beta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca²⁺ dependence of individual BK_Ca channels which we observed in electrophysiological recordings.

Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK_Ca, they express all three K_Ca types known. We suggest that the plurality and co-expression of different K_Ca channels and BK_Ca 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 K_Ca channel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.