Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC(3)H1 cells

Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC(3)H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ ''leak'' and enhances steady-state loading capacity of SR vesicles similar to 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [H-3]ryanodine (50% effective concentration of similar to 2 mu M). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry(1)R in intact BC(3)H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry(1)R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC(3)H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry(1)R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [H-3]ryanodine with high affinity.