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⁺]_i) 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_o 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 μM 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 PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.