Chu A, Díaz-Muñoz M, Hawkes M J, Brush K, Hamilton S L
Department of Medicine, Methodist Hospital, Houston, Texas.
Mol Pharmacol. 1990 May;37(5):735-41.
In this paper, we study the modulation of the rabbit fast twitch skeletal muscle calcium release channel by assaying the kinetics of [3H]ryanodine binding, 45Ca2+ flux, and single-channel activity. The effects of modulators of the Ca2+ release channel (confirmed here with both flux and single-channel data) were examined for effects on [3H]ryanodine binding to terminal cisternae vesicles. We find that activators of the release channel, such as adenine nucleotides (1 mM) and caffeine (1 mM), enhance the rate of association of [3H]ryanodine, whereas inhibitors, such as Mg2+ (1 mM) and ruthenium red (100 nM), decrease the rate of association. High concentrations of either ryanodine or ruthenium red, which close the channel, slow the dissociation of [3H]ryanodine, suggesting that at these concentrations the inhibitory effects of both ryanodine and ruthenium red occur as the result of binding at a site distinct from but interacting cooperatively with the high affinity site. Our data are consistent with a model in which the high affinity ryanodine binding site is within a conformationally sensitive area of the channel, such that conditions that open the channel (ATP, caffeine, etc.) enhance the rate at which [3H]ryanodine reaches its binding site and other conditions that close the channel (the binding of ryanodine and ruthenium red to a low affinity site) slow the dissociation of [3H]ryanodine from the high affinity site. Some conditions that inhibit channel activity (high concentrations of Mg2+ and Ca2+) slow association but do not affect dissociation of bound [3H]ryanodine, suggesting a completely different state of the channel from that which is inactive in the presence of high concentrations of ryanodine or ruthenium red. In summary, the functional state of the fast twitch skeletal muscle calcium release channel can be characterized by the changes in the kinetics of [3H]ryanodine binding. Different modulators (activators/inhibitors) affect different aspects of ryanodine binding (association/dissociation).
在本文中，我们通过测定[³H]ryanodine结合动力学、⁴⁵Ca²⁺通量和单通道活性，研究了兔快肌骨骼肌钙释放通道的调节作用。研究了钙释放通道调节剂（此处通量和单通道数据均证实）对[³H]ryanodine与终末池囊泡结合的影响。我们发现，释放通道的激活剂，如腺嘌呤核苷酸（1 mM）和咖啡因（1 mM），可提高[³H]ryanodine的结合速率，而抑制剂，如Mg²⁺（1 mM）和钌红（100 nM），则降低结合速率。高浓度的ryanodine或钌红会关闭通道，减缓[³H]ryanodine的解离，这表明在这些浓度下，ryanodine和钌红的抑制作用是由于它们在与高亲和力位点不同但协同相互作用的位点上结合所致。我们的数据与一个模型一致，即高亲和力ryanodine结合位点位于通道的构象敏感区域内，因此打开通道的条件（ATP、咖啡因等）会提高[³H]ryanodine到达其结合位点的速率，而关闭通道的其他条件（ryanodine和钌红与低亲和力位点的结合）会减缓[³H]ryanodine从高亲和力位点的解离。一些抑制通道活性的条件（高浓度的Mg²⁺和Ca²⁺）会减缓结合，但不影响结合的[³H]ryanodine的解离，这表明通道处于与高浓度ryanodine或钌红存在时的非活性状态完全不同的状态。总之，快肌骨骼肌钙释放通道的功能状态可以通过[³H]ryanodine结合动力学的变化来表征。不同的调节剂（激活剂/抑制剂）影响ryanodine结合的不同方面（结合/解离）。
