BASTADINS RELATE RYANODINE-SENSITIVE AND RYANODINE-INSENSITIVE CA2+ EFFLUX PATHWAYS IN SKELETAL SR AND BC(3)H1 CELLS

Bastadins are used to examine tile relationship between ryanodine-sens itive and ryanodine-insensitive Ca2+ efflux pathways that coexist in j unctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal mu scle and differentiated BC(3)H1 cells. Complete block of caffeine-sens itive Ca2+ channels with micromolar ryanodine or ruthenium red does no t alter the steady-state loading capacity of SR. Inhibition of sarco(e ndo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unma sks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Ba stadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryano dine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ '' leak'' and enhances steady-state loading capacity of SR vesicles simil ar to 2.5-fold. These actions of bastadins occur in the same concentra tion range that enhances the number of high-affinity binding sites for [H-3]ryanodine (50% effective concentration of similar to 2 mu M). Si milar 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 ryanodin e does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodin e nearly eliminates the Ca2+ leak unmasked by thapsigargin, even thoug h the Ca2+ stores are replete. The requirement of both a known Ry(1)R blocker and bastadins in combination provides a pharmacological link b etween ryanodine-sensitive Ca2+ channels and ryanodine-insensitive lea k pathways in isolated junctional SR and BC(3)H1 cells. Together, thes e results strongly suggest that bastadins, through their modulatory ac tions on the FKBP12-Ry(1)R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [H-3]ryanodin e with high affinity.