Intermediate Conductance Ca(2+)-Activated K(+) Channels Modulate Human Placental Trophoblast Syncytialization

Regulation of human placental syncytiotrophoblast renewal by cytotrophoblast migration, aggregation/fusion and differentiation is essential for successful pregnancy. In several tissues, these events are regulated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)), in part through their ability to regulate cell volume. We used cytotrophoblasts in primary culture to test the hypotheses that IK(Ca) participate in the formation of multinucleated syncytiotrophoblast and in syncytiotrophoblast volume homeostasis. Cytotrophoblasts were isolated from normal term placentas and cultured for 66 h. This preparation recreates syncytiotrophoblast formation in vivo, as mononucleate cells (15 h) fuse into multinucleate syncytia (66 h) concomitant with elevated secretion of human chorionic gonadotropin (hCG). Cells were treated with the IK(Ca) inhibitor TRAM-34 (10 µM) or activator DCEBIO (100 µM). Culture medium was collected to measure hCG secretion and cells fixed for immunofluorescence with anti-IK(Ca) and anti-desmoplakin antibodies to assess IK(Ca) expression and multinucleation respectively. K(+) channel activity was assessed by measuring (86)Rb efflux at 66 h. IK(Ca) immunostaining was evident in nucleus, cytoplasm and surface of mono- and multinucleate cells. DCEBIO increased (86)Rb efflux 8.3-fold above control and this was inhibited by TRAM-34 (85%; p<0.0001). Cytotrophoblast multinucleation increased 12-fold (p<0.05) and hCG secretion 20-fold (p<0.05), between 15 and 66 h. Compared to controls, DCEBIO reduced multinucleation by 42% (p<0.05) and hCG secretion by 80% (p<0.05). TRAM-34 alone did not affect cytotrophoblast multinucleation or hCG secretion. Hyposmotic solution increased (86)Rb efflux 3.8-fold (p<0.0001). This effect was dependent on extracellular Ca(2+), inhibited by TRAM-34 and 100 nM charybdotoxin (85% (p<0.0001) and 43% respectively) but unaffected by 100 nM apamin. In conclusion, IK(Ca) are expressed in cytotrophoblasts and their activation inhibits the formation of multinucleated cells in vitro. IK(Ca) are stimulated by syncytiotrophoblast swelling implicating a role in syncytiotrophoblast volume homeostasis. Inappropriate activation of IK(Ca) in pathophysiological conditions could compromise syncytiotrophoblast turnover and volume homeostasis in pregnancy disease.