title: Mechanism of Ca(2+)-dependent inactivation of L-type Ca2+ channels in GH3 cells: direct evidence against dephosphorylation by calcineurin.
abstract: Dephosphorylation of Ca2+ channels by the Ca(2+)-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca(2+)-dependent inactivation after photolysis. The results provide strong evidence against Ca(2+)-dependent dephosphorylation as the mechanism of Ca2+ current inactivation in GH3 cells, but support the alternative idea that Ca(2+)-dependent inactivation reflects a direct effect of intracellular Ca2+ on channel gating.
authors.affiliation: University of Texas Southwestern Medical Center, Molecular Cardiology Laboratories, Dallas, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Victor
authors.forename: F
authors.initials: F
authors.lastname: Rusnak
authors.forename: R
authors.initials: R
authors.lastname: Sikkink
authors.forename: E
authors.initials: E
authors.lastname: Marban
authors.forename: B
authors.initials: B
authors.lastname: O'Rourke
mesh_terms: D000818:Animals; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008954:Models, Biological; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D010782:Photolysis; D051381:Rats; D016559:Tacrolimus / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium; D016559:Tacrolimus