ALLOSTERIC SITE STRUCTURES OF CARDIOVASCULAR CHANNELS

心肌Ryanodine受体钙释放通道（RyR2）和平滑肌 大电导钙和电压激活钾（BK）通道是 正常和病理性心血管生理学。每一个都是成孔亚基的四聚体， 与辅助亚基复合，辅助亚基变构调节通道行为。FK506结合蛋白 （FKBP 12.6）调节RyR2，并且β 1调节BK通道α亚基。此外，这些渠道 通过具有治疗潜力的配体，如JTV519的RyR2，进行变构调节。了解 这些变构相互作用的机制，结合位点的氨基酸残基， FKBP12.6上和RyR2上的JTV519上和BK通道α亚基上的β 1亚基将被鉴定。 此外，将确定通道的不同功能状态下的接触的变化。 心肌肌浆网膜RyR2结合位点残基及其与BK通道的结合位点残基 在培养的细胞中异源表达的，将通过光亲和标记、化学标记和免疫荧光标记来标记。 交叉链接和足迹法。将通过蛋白裂解、分离和纯化来鉴定残基。 标记肽、质谱和微测序。此外，交联反应将 是指通过定点诱变在蛋白质上的特定位置取代的半胱氨酸残基。 靶蛋白，并通过Western印迹检测交联。修改 提出了已知的方法以使在低密度聚乙烯上的结合位点的详细表征变得可行。 丰度通道如BK和一个非常大的蛋白质如RyR2亚基。结果将 提供了对这两个通道中变构相互作用的分子机制的见解，并将 为靶向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.