Activation of a TRP-like channel and intracellular Ca²⁺ dynamics during phospholipase-C-mediated cell death

The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca²⁺ ([Ca²⁺]_c) dynamics and a distinct Ca²⁺ signature that involves Ca²⁺ influx from the external medium and internal Ca²⁺ stores. We investigated the molecular basis of this Ca²⁺ response by using [Ca²⁺]_c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca²⁺ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca²⁺ entry does not occur through the hitherto described high- and low-affinity Ca²⁺ uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP₃) receptor suggests that Ca²⁺ release from internal stores following IP₃ formation combines with the extracellular Ca²⁺ influx.