INTERDEPENDENCE OF RYANODINE BINDING, OLIGOMERIC RECEPTOR INTERACTIONS, AND CA2+ RELEASE REGULATION IN JUNCTIONAL SARCOPLASMIC-RETICULUM
INTERDEPENDENCE OF RYANODINE BINDING, OLIGOMERIC RECEPTOR INTERACTIONS, AND CA2+ RELEASE REGULATION IN JUNCTIONAL SARCOPLASMIC-RETICULUM
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DOI：
10.1016/0003-9861(91)90615-p
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发表时间：
1991-10-01
影响因子：
3.9
通讯作者：
INESI, G
中科院分区：
文献类型：
--
作者：
CARROLL, S;SKARMETA, JG;INESI, G
我们研究了兰尼碱结合其受体（RR），并比较其对Ca2+释放的影响，由Ca2+加ATP触发的Ca2+释放，使用囊泡片段的连接末端池（JTC）从骨骼肌。Ryanodine结合缓慢（需要数小时或数天才能完成），并且高度依赖于温度（Q10= 4）和Ca2+。在平衡状态下，当兰尼碱浓度超过10− 9 m时，结合的程度增加，表现出负协同效应，并在10− 5 m兰尼碱附近达到560，000 Da RR链的化学计量。竞争性结合的兰尼碱类似物证明了高亲和力结合的特异性。使用快速过滤的动力学研究表明，在没有兰尼定的情况下，在加载的JTC囊泡触发暴露于含有Ca2+加ATP的灌注介质后，Ca2+快速（k= 15 s−1）释放。这种释放的诱导没有滞后期，并显示出最小的温度依赖性。相反，JTC囊泡长时间暴露于低浓度（10− 7 m）的兰尼碱会使JTC变成一种允许缓慢（k= 1 s−1）释放Ca2+的状态，即使在没有Ca2++ ATP触发的情况下也是如此。更高浓度（>μm）的兰尼碱不允许任何Ca2+释放，甚至阻止通常由Ca2+加ATP触发的释放。我们的数据表明，ryanodine结合到开放状态的四聚体RR，诱导蛋白质构象的变化和改变寡聚体的相互作用。第一个分子的Ryanodine的受体上的四个结合位点之一的结合产生部分关闭和低电导状态的Ca2+释放通道，并降低其余网站的Ryanodine结合亲和力。Ryanodine占据受体上的所有四个结合位点，完成Ca2+通道的关闭，并阻断Ca2+加ATP的触发作用。RR链的四聚体缔合通过与双功能试剂交联来证明，产生保留兰尼碱结合和Ca2+释放功能的交联四聚体。
We have examined ryanodine binding to its receptor (RR) and compared its effect on Ca2+release to the Ca2+release triggered by Ca2+plus ATP, using vesicular fragments of junctional terminal cisternae (JTC) obtained from skeletal muscle. Ryanodine binding is slow (taking hours or days to complete) and is highly temperature (Q10= 4) and Ca2+dependent. At equilibrium, the extent of binding increases as the concentration of ryanodine is raised above 10−9m, exhibiting negative cooperativity and reaching the stoichiometry of the 560,000-Da RR chains near 10−5mryanodine. The specificity of the high affinity binding is demonstrated by competitive binding of ryanodine analogs. Kinetic studies using rapid filtration show that, in the absence of ryanodine, rapid (k= 15 s−1) release of Ca2+follows a triggering exposure of loaded JTC vesicles to perfusion media containing Ca2+plus ATP. Induction of this release has no lag period and displays minimal temperature dependence. In contrast, prolonged exposure of JTC vesicles to low (10−7m) ryanodine concentrations changes the JTC to a state permitting slow (k= 1 s−1) release of Ca2+even in the absence of the Ca2+plus ATP trigger. Higher (>μm) concentrations of ryanodine do not allow any Ca2+release and prevent even the release normally triggered by Ca2+plus ATP. Our data suggest that ryanodine binds to the open state of the tetrameric RR, inducing protein conformational changes and altered oligomeric interactions. Binding of the first molecule of ryanodine to one of the four binding sites on the receptor produces a partially closed and low conductance state of the Ca2+release channel and reduces the ryanodine binding affinity of the remaining sites. Ryanodine occupancy of all four binding sites on the receptor completes closure of the Ca2+channel and blocks the triggering action of Ca2+plus ATP. The tetrameric association of the RR chains is demonstrated by crosslinking with bifunctional reagents, generating crosslinked tetramers that retain ryanodine binding and Ca2+release functions.