Role of store-dependent influx of Ca2+ and efflux of K+ in apoptosis of CHO cells.

Agents mobilising Ca(2+) from the endoplasmic reticulum are known to activate apoptosis. Whatever means are used, the release of Ca(2+) is often followed by a store-dependent entry of Ca(2+). Whether apoptosis is triggered by the depletion of the stores or by the subsequent store-dependent entry of Ca(2+) is still a matter of controversy. Here we studied apoptosis in CHO cells transfected with the rat neurotensin (NT) receptor, in which the store-dependent entry of Ca(2+) is abolished by repressing the transient receptor potential channel 2 (TRPC2) by an antisense oligonucleotide strategy (TRPC2(-) cells) [Cell Calcium 30 (2001) 157]. When stimulated with thapsigargin (TG), apoptosis occurred in both TRPC2(+) and TRPC2(-) cells but 12h earlier in TRPC2(+) cells, suggesting that store-dependent entry of Ca(2+) can accelerate the process. The expression and localisation of caspase-12, an enzyme that has been involved in the apoptosis triggered by a stress on the endoplasmic reticulum, was not different in TRPC2(+) and TRPC2(-) cells. On the contrary, the expression of GADD153 (Growth Arrest and DNA Damage inducible gene 153) triggered by TG treatment depended on external Ca(2+) and occurred earlier in TRPC2(+) than in TRPC2(-) cells. In these cells, we also noted the presence of K(+) channels activated by Ca(2+) (K(Ca) channels). Stimulation of TRPC2(+) cells with TG or with NT triggered a long sustained K(+) current, parallel to [Ca(2+)](i) transients, and resulting in a sustained hyperpolarisation of the cell membrane. K(+) current and hyperpolarisation were transient and not sustained in TRPC2(-) cells. Inhibition of K(Ca) channels with charybdotoxin dramatically reduced the K(+) current and also significantly brought down the level of apoptosis, suggesting that a prolonged efflux of K(+) could be involved in the apoptosis process. We conclude that in CHO cells, store-dependent entry of Ca(2+) can accelerate apoptosis by accelerating the expression of GADD153 and by inducing a prolonged efflux of K(+) out of the cell.