Protein kinase C activates store-operated Ca(2+) channels in human glomerular mesangial cells.
Store-operated Ca(2+) 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 Ca(2+) concentration ([Ca(2+)](i)) revealed that the thapsigargin-induced Ca(2+) entry pathway was abolished by calphostin C, a protein kinase C inhibitor. The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca(2+) influx that was significantly attenuated by calphostin C and La(3+) but not by diltiazem. Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca(2+)](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 [Ca(2+)](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 microm 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 [Ca(2+)] did not significantly affect the channel activity in inside-out patch. These results indicate that the depletion of Ca(2+) stores activates SOC by PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.
Ma
R
R
Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575, USA.
Pluznick
J
J
Kudlacek
P
P
Sansom
S C
SC
D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D005920:Glomerular Mesangium / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D017136:Ion Transport / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology
D015220:Calcium Channels; D011493:Protein Kinase C; D002118:Calcium