Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells.
Protein kinase C (PKC) plays an important role in activating store-operated Ca2+ channels (SOC) in human mesangial cells (MC). The present study was performed to determine the specific isoform(s) of conventional PKC involved in activating SOC in MC. Fura 2 fluorescence ratiometry showed that the thapsigargin-induced Ca2+ entry (equivalent to SOC) was significantly inhibited by 1 microM Gö-6976 (a specific PKCalpha and betaI inhibitor) and PKCalpha antisense treatment (2.5 nM for 24-48 h). However, LY-379196 (PKCbeta inhibitor) and 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether (HBDDE; PKCalpha and gamma inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKCalpha antisense significantly depressed thapsigargin-induced activation of SOC. However, LY-379196 and HBDDE did not affect the SOC responses. In inside-out patches, application of purified PKCalpha or betaI, but not betaII or gamma, significantly rescued SOC from postexcision rundown. Western blot analysis revealed that thapsigargin evoked a decrease in cytosolic expression with a corresponding increase in membrane expression of PKCalpha and gamma. However, the translocation from cytosol to membranes was not detected for PKCbetaI or betaII. These results suggest that PKCalpha participates in the intracellular signaling pathway for activating SOC upon release of intracellular stores of Ca2+.
Ma
Rong
R
Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575, USA.
Kudlacek
Patrick E
PE
Sansom
Steven C
SC
D000906:Antibodies / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D025802:Electric Capacitance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D019284:Thapsigargin / Q000494:pharmacology
D000906:Antibodies; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D019284:Thapsigargin; C087857:protein kinase C gamma; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D016257:Fura-2