RYANODINE RECEPTORS AND ACCESSORY PROTEINS

摘要：肌浆网钙释放通道/兰尼定受体(RyR)是心肌细胞“钙释放单位”的主要组成部分。其他胞质辅助蛋白包括钙调蛋白、FK506结合蛋白和山梨素。在通道的管腔一侧，钙离子通道蛋白与钙离子结合，并与RyRs紧密结合。因此，心肌细胞的钙释放单位是一个异源系统，包括RyR同源四聚体和几种胞浆和管腔蛋白。辅助蛋白的作用以及在E-C偶联过程中完全整合的钙释放单位的功能输出仍然不完全清楚。这项建议将剖析RyRs的每一种辅助蛋白对心脏细胞内钙释放的贡献。我们认为，RyRs的辅助蛋白是心脏钙释放单位不可或缺的元素，使RyRs能够抵消钙诱导的钙释放(CICR)固有的正反馈。为了验证这一假说，我们建议：1)定义纯化的(“裸”)RyR的钙反应。无辅助蛋白的RyRs将在脂质双层中重组，并被快速的[Ca~(2+)]刺激激活，类似于外部Ca~(2+)通过肌膜通道的内流，从而提供RyR对Ca~(2+)的内在反应的完整轮廓。2)确定天然RyRs和含有辅助蛋白的纯化RyRs的钙反应动力学。如上所述，将获得天然(SR包埋)RyR的Po-[Ca~(2+)]关系，并与在CaM、FKBP和山梨素选择性存在下纯化的RyR所获得的关系进行比较。3)测定磷酸化/去磷酸化对钙释放单位对钙反应的影响。RyRs的磷酸化调节心脏的钙释放。我们将确定PKA和CaMKII对纯化的、天然的RyR和受个别辅助蛋白调控的RyR的钙反应动力学的影响。这个日益复杂的系统，从单一的RyR通道到协同工作的分子集合，将定义心肌中“钙释放单位”的动力学特性和调节机制。由此获得的结果将有助于解决该单位的个别成分对完整心肌细胞内钙瞬变的影响。

Abstract: The sarcoplasmic reticulum Ca2+ release channel/ryanodine receptor (RyR), is the main component of the "Ca2+ release unit" of cardiac cells. Other cytosolic accessory proteins include calmodulin, FK506 binding protein and sorcin.. In the lumenal side of the channel, calsequestrin binds Ca2+ and is firmly associated to RyRs. The Ca2+ release unit of cardiac cells is therefore a heterologous system that encompasses the RyR homotetramer and several cytosolic and lumenal proteins. The role of accessory proteins and, consequently, the functional output of a fully- integrated Ca2+ release unit during E-C coupling remain incompletely understood. This proposal will dissect the contribution of each of the accessory proteins of RyRs to intracellular Ca2+ release in the heart. We propose that accessory proteins of RyRs are indispensable elements of the Ca2+ release unit of the heart that allow RyRs to counteract the inherently positive feedback of Ca2+-induced Ca2+ release (CICR). To test this hypothesis, we propose: 1) To define the Ca2+ response of purified ("naked") RyRs. Accessory protein-free RyRs will be reconstituted in lipid bilayers and activated by fast [Ca2+] stimuli that resembles the influx of external Ca2+ through sarcolemmal channels, thus providing an integral outline of the intrinsic response of RyRs to Ca2+. 2) To define the kinetics of Ca2+ response of native RyRs, and of purified RyRs in the presence of accessory proteins. Po-[Ca2+] relationships will be obtained for native (SR-embedded) RyR as above, and compared to those obtained with purified RyR in the selective presence of CaM, FKBP, and sorcin. 3) To determine the effect of phosphorylation/dephosphorylation on the response of the Ca2+ release unit to Ca2+. Phosphorylation of RyRs modulate the Ca2+ release in heart. We will determine the effect of PKA and CaMKII on the kinetics of the Ca2+ response of purified, native RyR, and RyRs regulated by individual accessory proteins. This system of increasing complexity that ranges from a single RyR channel to an ensemble of molecules working in unison will define the kinetic properties and mechanisms of regulation of the "Ca2+ release unit" in heart muscle. Results thus obtained will help resolve the effect of individual components of this unit on the intracellular Ca2+ transient of intact cardiac cells.