ALLOSTERIC SITE STRUCTURES OF CARDIOVASCULAR CHANNELS

心肌Ryanodine受体钙释放通道(RyR2)与平滑肌 大电导钙和电压激活的钾通道(BK)是重要的信号转导途径 正常和病理的心血管生理学。每一个都是一个四聚体，在一个孔形成亚基 具有变构调节通道行为的辅助亚单位的复合体。FK506结合蛋白 (FKBP12.6)调制RyR2，并且Betal调制BK通道Alpha Subunit。另外，这些渠道 受具有治疗潜力的配体的变构调节，如JTV519的RyR2。要理解 这些变构相互作用的机制，结合部位的氨基酸残基 将识别FKBP12.6和RyR2上的JTV519以及BK通道上的β亚基。 此外，将确定在通道的不同功能状态下触点的变化。 RyR2在心肌肌浆网膜、RyR2和BK通道中的结合位点残基 在培养细胞中异源表达，将通过光亲和标记，化学 交叉链接和足迹方法。残留物将通过蛋白质裂解、分离 标记肽、质谱学和显微测序。此外，交联反应将 针对半胱氨酸残基，在特定位置进行定点突变 目的蛋白，并通过Western blotting检测到交联物。修改 已知的方法被提出以使低结合部位的详细表征成为可能 像BK一样的丰度通道，以及像RyR2亚单位这样的非常大的蛋白质。结果将会是 洞察这两个通道中变构相互作用的分子机制，并将 为针对这些通道的心血管治疗提供分子结构基础。

The cardiac ryanodine receptor calcium-release channel (RyR2) and the smooth-muscle large-conductance calcium-and-voltage-activated potassium (BK) channel are vital participants in normal and pathological cardiovascular physiology. Each is a tetramer of pore-forming subunits in a complex with auxiliary subunits that allosterically modulate channel behavior. FK506-binding protein (FKBP12.6) modulates RyR2, and betal modulates BK channel alpha subu'nit. In addition, these channels are allosterically modulated by ligands with therapeutic potential, such as RyR2 by JTV519. To understand the mechanisms of these allosteric interactions, the amino acid residues in the binding sites for FKBP12.6 and for JTV519 on RyR2 and for betal subunit on BK channel alpha subunit will be identified. Furthermore, changes in the contacts in different functional states of the channels will be determined. Binding-site residues in RyR2 in cardiac sarcoplasmic reticulum membrane, and in RyR2 and BK channel heterologously expressed in cultured cells, will be tagged by photoaffinity labeling, chemical crbsslinking, and foot-printing methods. The residues will be identified by protein cleavage, isolation of labeled peptides, mass spectrometry and micro-sequencing. In addition, cross-linking reactions will be directed to cysteine residues substituted by site-directed mutagenesis at specific locations on the target proteins, and cross-linking will be detected by Western blotting. Modifications of known methods are proposed to make feasible the detailed characterization of binding sites on a low abundance channel like BK and on a very large protein like the RyR2 subunit. The results will provide insights into the molecular mechanisms of allosteric interactions in these two channels and will provide molecular structural bases for cardiovascular therapeutics targeted at these channels.