Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence... Dephosphorylation of Ca2+ channels by the Ca2+-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca2+-dependent inactivation of Ca2+ current in rat pituitary tumor (GH3) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca2+ to the channel protein, the alternative ``calcineurin hypothesis'' has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH3 cells. To determine if calcineurin plays a part in the voltage- and/or Ca2+-dependent components of dihydropyridine-sensitive Ca2+ current decay, we rapidly altered the intracellular Ca2+ buffering capacity of GH3 cells 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 for Ca2+. Despite confirmation of the abundance of calcineurin in the GH3 cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca2+-dependent inactivation of Ca2+ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca2+-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca2+-dependent inactivation after photolysis. The results provide strong evidence against Ca2+-dependent dephosphorylation as the mechanism of Ca2+ current inactivation in GH3 cells, but support the alternative idea that Ca2+-dependent inactivation reflects a direct effect of intracellular Ca2+ on channel gating. The Journal of Membrane Biology Springer Journals

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

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Copyright © Inc. by 1997 Springer-Verlag New York
Life Sciences; Biochemistry, general; Human Physiology
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