RYANODINE RECEPTORS AND ACCESSORY PROTEINS

摘要：肌浆网Ca2+释放通道/ryanodine受体（RyR）是心肌细胞“Ca2+释放单元”的主要组成部分。其他细胞质辅助蛋白包括钙调蛋白、FK506结合蛋白和sorcin。在通道的管腔侧，钙调钙素结合Ca2+并与RyRs紧密相关。因此，心肌细胞的Ca2+释放单元是一个包含RyR四聚体和几种细胞质和管腔蛋白的异源系统。辅助蛋白的作用，因此，在E-C偶联过程中，一个完全集成的Ca2+释放单元的功能输出仍然不完全清楚。本提案将剖析RyRs的每一个辅助蛋白对心脏细胞内Ca2+释放的贡献。我们认为RyRs的辅助蛋白是心脏Ca2+释放单元不可或缺的元素，允许RyRs抵消Ca2+诱导的Ca2+释放（CICR）固有的正反馈。为了验证这一假设，我们提出：1)定义纯化（“裸”）RyRs的Ca2+响应。辅助无蛋白的RyRs将在脂质双分子层中重建，并被快速[Ca2+]刺激激活，类似于通过肌层通道流入的外部Ca2+，从而提供RyRs对Ca2+的内在反应的整体轮廓。2)定义原生RyRs和纯化RyRs在辅助蛋白存在下的Ca2+响应动力学。如上所述，将获得天然（sr嵌入）RyR的Po-[Ca2+]关系，并与在CaM， FKBP和sorcin选择性存在下纯化RyR的关系进行比较。3)确定磷酸化/去磷酸化对Ca2+释放单元对Ca2+响应的影响。RyRs磷酸化调节心脏Ca2+释放。我们将确定PKA和CaMKII对纯化的、天然的RyR和由单个辅助蛋白调节的RyR的Ca2+响应动力学的影响。从单一的RyR通道到协同工作的分子集合，这个日益复杂的系统将定义心肌中“Ca2+释放单元”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元的单个组分对完整心脏细胞内Ca2+瞬态的影响。

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.