Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on I(CRAC)-like calcium channels

Activation of phospholipase C in nonexcitable cells causes the release of calcium (Ca(2+)) from intracellular stores and activation of Ca(2+) influx by means of Ca(2+) 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 Ca(2+) channels in human carcinoma A431 cells, which can be activated by extracellular UTP, by depletion of intracellular Ca(2+) stores after exposure to the Ca(2+)-pump inhibitor thapsigargin, or by loading the cells with Ca(2+) 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 Ca(2+) 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) Ca(2+) channels by extracellular UTP and intracellular inositol (1,4,5)-triphosphate (IP(3)), apparently because of reduction in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels in the patch. Prolonged exposure of patches to thapsigargin renders I(CRACL) Ca(2+) channels unresponsive to IP(3) but still available to activation by the combined action of IP(3) and anti-PIP(2) antibody. Based on these data, we concluded that phospholipase C-mediated and store-operated Ca(2+) influx pathways in A431 cells converge on the same I(CRACL) Ca(2+) channel, which can be modulated by PIP(2).