Pessah I N, Molinski T F, Meloy T D, Wong P, Buck E D, Allen P D, Mohr F C, Mack M M
Department of Molecular Biosciences, University of California, Davis 95616, USA.
Am J Physiol. 1997 Feb;272(2 Pt 1):C601-14. doi: 10.1152/ajpcell.1997.272.2.C601.
Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). 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 BC3H1 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 approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 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 Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.
巴斯他汀能与骨骼肌中12 kDa的FK-506结合蛋白（FKBP12）-雷诺丁受体（Ry1R）复合物发生强力相互作用，以增强高亲和力的雷诺丁结合构象（M. M. 麦克、T. F. 莫林斯基、E. D. 巴克和I. N. 佩萨。《生物化学杂志》269: 23236 - 23249, 1994）。巴斯他汀用于研究在兔骨骼肌和分化的BC3H1细胞的连接肌浆网（SR）囊泡中共存的雷诺丁敏感和雷诺丁不敏感的Ca²⁺外流途径之间的关系。用微摩尔浓度的雷诺丁或钌红完全阻断咖啡因敏感的Ca²⁺通道，不会改变SR的稳态负载能力。用毒胡萝卜素抑制肌浆内质网Ca²⁺-ATP酶（SERCA）泵，会暴露出一条对雷诺丁和钌红不敏感的Ca²⁺外流途径。单独使用巴斯他汀5不会抑制因SERCA泵抑制而暴露的Ca²⁺外流，但与阻断浓度的雷诺丁或钌红联合使用时，它能消除雷诺丁不敏感的Ca²⁺“渗漏”，并使SR囊泡的稳态负载能力提高约2.5倍。巴斯他汀的这些作用发生在增强[³H]雷诺丁高亲和力结合位点数量的相同浓度范围内（50%有效浓度约为2 μM）。FK-506和雷诺丁联合使用对SR Ca²⁺转运有类似作用。用雷诺丁阻断完整BC3H1细胞中的Ry1R，不会消除因毒胡萝卜素暴露的显著Ca²⁺渗漏。即使Ca²⁺储存充足，巴斯他汀与雷诺丁的膜通透性混合物联合使用几乎能消除因毒胡萝卜素暴露的Ca²⁺渗漏。已知的Ry1R阻断剂和巴斯他汀联合使用的必要性，为分离的连接SR和BC3H1细胞中雷诺丁敏感Ca²⁺通道和雷诺丁不敏感渗漏途径之间提供了药理学联系。总之，这些结果强烈表明，巴斯他汀通过对FKBP12-Ry1R复合物的调节作用，将雷诺丁不敏感的渗漏状态转化为能以高亲和力识别[³H]雷诺丁的雷诺丁敏感通道。
