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 (Ca²⁺) from intracellular stores and activation of Ca²⁺ influx by means of Ca²⁺ release-activated channel (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 be modulated by PIP₂.