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 (I-CRAC) in the plasma membrane. The molecular identity and the mechanism of I-CRAC 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 I-CRAC channels. Thus, we concluded that the depletion-activated Ca2+ current in A431 cells is supported by I-CRAC-like (I-CRACL) channels, identical to I-min. We further demonstrated synergism in activation of I-CRACL 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 I-CRACL 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 he modulated by PIP2.