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

Store-operated Ca2+ channels (SOC) are expressed in cultured human mesangia l cells and activated by epidermal growth factor through a pathway involvin g protein kinase C (PKC), We used fura-2 fluorescence and patch clamp exper iments to determine the role of PKC in mediating the activation of SOC afte r depletion of internal stores by thapsigargin, The measurements of intrace llular Ca2+ concentration ([Ca2+],) revealed that the thapsigargin-induced Ca2+ entry pathway was abolished by calphostin C, a protein kinase C inhibi tor, The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a C a2+ influx that was significantly attenuated by calphostin C and La3+ but n ot by diltiazem, Neither PMA nor calphostin C altered the thapsigargin-indu ced initial transient rise in [Ca2+](i-). In cell-attached patch clamp expe riments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaf fected by thapsigargin when clamping [Ca2+](i) with 1,2-bis (o-Aminophenoxy )ethane-N,N,N ' ,N ' tetraacetic acid tetra(acetoxymethyl)ester. In the abs ence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significa nt increase in NP, of SOC, whereas calphostin C did not affect base-line ch annel activity, In inside-out patches, SOC activity ran down immediately up on excision but was reactivated significantly after adding the catalytic su bunit of 0.1 unit/ml of PKC plus 100 mum ATP, Neither ATP alone nor ATP wit h heat-inactivated PKC rescued a rundown of SOC, Metavanadate, a general pr otein phosphatase inhibitor, also enhanced SOC activity in inside-out patch es. Bath [Ca2+] did not significantly affect the channel activity in inside -out patch, These results indicate that the depletion of Ca2+ stores activa tes SOC by PRC-mediated phosphorylation of the channel proteins or a membra ne-associated complex.