Damiani E, Margreth A
Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Italia.
J Muscle Res Cell Motil. 1994 Apr;15(2):86-101. doi: 10.1007/BF00130421.
We have investigated high-affinity ryanodine-binding sites in membrane preparations from representative fast-twitch and slow-twitch muscles of the rabbit and rat, as well as from human mixed muscle. Our results, obtained in high-ionic strength binding buffer, demonstrate extensive similarities in binding affinity for [3H]ryanodine (Kd: about 10 nM) and a two-fold to four-fold difference in membrane density of the ryanodine receptor between fast-twitch and slow-twitch muscle of the rat and rabbit, respectively. The [3H]ryanodine-pCa relationship for the Ca(2+)-activation curve of ryanodine binding was found to be similar for all mammalian muscles, as tested at 20 nM ryanodine. With 10 mM caffeine or 50 microM doxorubicin the pCa for half-maximal activation of [3H]ryanodine binding invariably shifted from an average pCa value of 6.5 to pCa 7.1-7.3. IC50 values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca(2+)-release channel blocker, did not differ significantly (range 0.3-1.0 microM). The Ca(2+)-dependence curve (range 1 nM-10 mM free Ca2+) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cisternae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cisternae from the same species. Cardiac ryanodine receptor is also clearly distinguishable for electrophoretic mobility, Cleveland's peptide maps, and, most strikingly, for total lack of cross-reactivity with polyclonal antibody to fast skeletal RyR. By the same properties, the ryanodine receptor of fast- and slow-twitch muscle appear to be the same or a similar protein. On investigating the composition of calsequestrin in rat and human skeletal muscles, both in membrane-bound form and after purification by phenyl-Sepharose chromatography, we have been able to show that, independent of the animal species, the cardiac isoform, as characterized by the identical amino-terminal amino-acid sequence, pattern of immunoreactivity, and lack of Ca(2+)-dependent shift in mobility on SDS-PAGE, is exclusively expressed in slow-twitch fibres, together with the main fast-skeletal calsequestrin isoform. While our experimental findings strongly argue for the presence of only one population of skeletal-specific Ca(2+)-release channels in junctional terminal cisternae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of Ca(2+)-release, depending on its isoform composition.
我们研究了来自兔和大鼠典型快肌与慢肌以及人类混合肌的膜制剂中的高亲和力ryanodine结合位点。我们在高离子强度结合缓冲液中获得的结果表明，对[³H]ryanodine的结合亲和力（Kd：约10 nM）存在广泛相似性，并且大鼠和兔的快肌与慢肌之间ryanodine受体的膜密度分别存在2至4倍的差异。在20 nM ryanodine下测试时，发现所有哺乳动物肌肉中ryanodine结合的Ca²⁺激活曲线的[³H]ryanodine-pCa关系相似。使用10 mM咖啡因或50 μM阿霉素时，[³H]ryanodine结合半最大激活的pCa始终从平均pCa值6.5变为pCa 7.1 - 7.3。Ca²⁺释放通道阻滞剂钌红对[³H]ryanodine结合的抑制IC50值没有显著差异（范围为0.3 - 1.0 μM）。我们在5 nM ryanodine下观察到的兔快肌和慢肌终末池[³H]ryanodine结合的Ca²⁺依赖性曲线（游离Ca²⁺范围为1 nM - 10 mM）呈钟形，与同一物种心脏终末池获得的曲线不同。心脏ryanodine受体在电泳迁移率、Cleveland肽图方面也明显可区分，最显著的是与抗快肌骨骼肌RyR多克隆抗体完全缺乏交叉反应性。基于相同的特性，快肌和慢肌的ryanodine受体似乎是相同或相似的蛋白质。在研究大鼠和人类骨骼肌中钙结合蛋白的组成时，无论是膜结合形式还是通过苯基琼脂糖凝胶色谱纯化后，我们都能够表明，与动物物种无关，以相同的氨基末端氨基酸序列、免疫反应模式以及SDS - PAGE上缺乏Ca²⁺依赖性迁移变化为特征的心脏同工型仅在慢肌纤维中表达，同时还表达主要的快肌骨骼肌钙结合蛋白同工型。虽然我们的实验结果有力地支持了哺乳动物快肌和慢肌连接终末池中仅存在一种骨骼肌特异性Ca²⁺释放通道的观点，但同时也表明根据其同工型组成，钙结合蛋白对Ca²⁺释放的调节存在差异。
