Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on ICRAC-like calcium channels.
Activation of phospholipase C in nonexcitable cells causes the release of calcium (Ca2+) from intracellular stores and activation of Ca2+ influx by means of Ca2+ release-activated channels (ICRAC) in the plasma membrane. The molecular identity and the mechanism of ICRAC channel activation are poorly understood. Using the patch-clamp technique, here we describe the plasma membrane Ca2+ channels in human carcinoma A431 cells, which can be activated by extracellular UTP, by depletion of intracellular Ca2+ stores after exposure to the Ca2+-pump inhibitor thapsigargin, or by loading the cells with Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. The observed channels display the same conductance and gating properties as previously described I(min) channels, but have significantly lower conductance for monovalent cations than the ICRAC channels. Thus, we concluded that the depletion-activated Ca2+ current in A431 cells is supported by I(CRAC)-like (ICRACL) channels, identical to I(min). We further demonstrated synergism in activation of ICRACL Ca2+ channels by extracellular UTP and intracellular inositol (1,4,5)-triphosphate (IP3), apparently because of reduction in phosphatidylinositol 4,5-bisphosphate (PIP2) levels in the patch. Prolonged exposure of patches to thapsigargin renders ICRACL Ca2+ channels unresponsive to IP3 but still available to activation by the combined action of IP3 and anti-PIP2 antibody. Based on these data, we concluded that phospholipase C-mediated and store-operated Ca2+ influx pathways in A431 cells converge on the same I(CRACL) Ca2+ channel, which can be modulated by PIP2.
Kaznacheyeva
E
E
Institute of Cytology RAS, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia.
Zubov
A
A
Gusev
K
K
Bezprozvanny
I
I
Mozhayeva
G N
GN
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002277:Carcinoma / Q000201:enzymology / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004553:Electric Conductivity; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
D015220:Calcium Channels; D019269:Phosphatidylinositol 4,5-Diphosphate; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium; D014544:Uridine Triphosphate