MECHANISM OF CA-2-DEPENDENT INACTIVATION OF L-TYPE CA2+ CHANNELS IN GH(3) CELLS - DIRECT EVIDENCE AGAINST DEPHOSPHORYLATION BY CALCINEURIN()

Dephosphorylation of Ca2+ channels by the Ca2+-activated phosphatase 2 B (calcineurin) has been previously suggested as a mechanism of Ca2+-d ependent inactivation of Ca2+ current in rat pituitary tumor (GH(3)) c ells. Although recent evidence favors an inactivation mechanism involv ing direct binding of Ca2+ to the channel protein, the alternative ''c alcineurin hypothesis'' has not been critically tested using the speci fic calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH, cells. To determine if calcineurin plays a part in the voltage-and/or Ca2+-d ependent components of dihydropyridine-sensitive Ca2+ current decay, w e rapidly altered the intracellular Ca2+ buffering capacity of GH, cel ls by flash photolysis of DM-nitrophen, a high affinity Ca2+ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca2+ current inactivation in a two-pulse voltage protocol with Ca2 + as the charge carrier, but had no effect when Ba2+ was substituted f or Ca2+. Despite confirmation of the abundance of calcineurin in the G H, cells by biochemical assays, acute application of CsA or FK506 afte r photolysis had no effect on Ca2+-dependent inactivation of Ca2+ curr ent, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK 506 or CsA did not inhibit Ca2+-dependent inactivation. Similarly, blo cking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca2+-dependent inact ivation after photolysis. The results provide strong evidence against Ca2+-dependent dephosphorylation as the mechanism of Ca2+ current inac tivation in GH(3) cells, but support the alternative idea that Ca2+-de pendent inactivation reflects a direct effect of intracellular Ca2+ on channel gating.