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

ABSTRACT: BACKGROUND: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. METHODS: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. RESULTS: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. CONCLUSION: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.

SUBMITTER: Traut MH

PROVIDER: S-EPMC2671515 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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

Traut Matthias H MH Berg Dieter D Berg Ulrike U Mayerhofer Artur A Kunz Lars L

Reproductive biology and endocrinology : RB&E 20090408

<h4>Background</h4>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-s ...[more]

PMID: 19351419

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Project description:Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.

Dataset Information

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

Publications

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

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