Protein Kinase C Activates Store-operated Ca2+ Channels in Human Glomerular Mesangial Cells

Rong Ma, Jennifer Pluznick, Patrick Kudlacek, Steven C. Sansom

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

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 NPo 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.

Original languageEnglish
Pages (from-to)25759-25765
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
StatePublished - 13 Jul 2001

Fingerprint Dive into the research topics of 'Protein Kinase C Activates Store-operated Ca<sup>2+</sup> Channels in Human Glomerular Mesangial Cells'. Together they form a unique fingerprint.

  • Cite this