Protein kinase C activates store-operated Ca2+ channels in human glomerular mesangial cells

Store-operated Ca2+ channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC), We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin, The measurements of intracellular Ca2+ concentration ([Ca2+],) revealed that the thapsigargin-induced Ca2+ entry pathway was abolished by calphostin C, a protein kinase C inhibitor, The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca2+ influx that was significantly attenuated by calphostin C and La3+ but not by diltiazem, Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca2+](i-). In cell-attached patch clamp experiments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaffected by thapsigargin when clamping [Ca2+](i) with 1,2-bis (o-Aminophenoxy)ethane-N,N,N ' ,N ' tetraacetic acid tetra(acetoxymethyl)ester. In the absence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significant increase in NP, of SOC, whereas calphostin C did not affect base-line channel activity, In inside-out patches, SOC activity ran down immediately upon excision but was reactivated significantly after adding the catalytic subunit of 0.1 unit/ml of PKC plus 100 mum ATP, Neither ATP alone nor ATP with heat-inactivated PKC rescued a rundown of SOC, Metavanadate, a general protein phosphatase inhibitor, also enhanced SOC activity in inside-out patches. Bath [Ca2+] did not significantly affect the channel activity in inside-out patch, These results indicate that the depletion of Ca2+ stores activates SOC by PRC-mediated phosphorylation of the channel proteins or a membrane-associated complex.