STRUCTURAL ANALYSIS OF THE CA++ RELEASE CHANNEL

骨骼肌Ca 2+释放通道或ryanodine受体（RYR 1）跨膜区中孔的开放受多种调节剂与其胞质结构域结合的调节。 产生两种人类疾病，中央核心疾病（CCD）和恶性高热（MH）的突变也经常在胞质结构域中发现。 本申请的总体目标是阐明细胞质调节剂的结合或突变的存在改变通道门控的分子机制。 本申请的工作假设是调节剂结合接近并调节变构调节跨膜结构域中孔门控的亚基内或亚基间接触位点。 我们还提出，两个细胞质结构域的恶性高热和中央核心疾病突变发生相互作用，形成一个红外或intersubunit接触网站接近FKBP 12的结合位点，在任何一个相互作用的合作伙伴的突变改变调制器结合和intersubunit通信。 具体目标是：1）描绘开放和闭合构象中通道之间的差异，以鉴定通道门控中的结构步骤，2）评估调节剂对RYR 1结构的贡献，3）定位RYR 1的3D结构中的MH/CCD突变位点和半胱氨酸3635，并确认蛋白质-蛋白质相互作用。将使用冷冻电子显微镜和计算机重建获得结构信息。 结构研究将通过功能分析进行验证，功能分析使用重构为平面脂质双层的通道的单通道分析和[3 H]ryanodine结合分析。 RYR 1内的相互作用结构域将通过使用生物传感器测定评估表达片段相互作用的能力和通过使用酵母相互作用陷阱鉴定相互作用区域来鉴定。

The opening of the pore in the transmembrane regions of the skeletal muscle Ca2+ release channel or ryanodine receptor (RYR1) is regulated by the binding of a variety of modulators to its cytoplasmic domains. Mutations that produce the two human diseases, central core disease (CCD) and malignant hyperthermia (MH), are also found frequently in cytoplasmic domains. The overall objective of this application is the elucidation of the molecular mechanisms by which binding of cytoplasmic modulators or the presence of mutations alter channel gating. The working hypothesis for this application is that the modulators bind close to and modulate infra or intersubunit contact sites that allosterically regulate the gating of the pore in the transmembrane domain. We also propose that the two cytoplasmic domains where the malignant hyperthermia and central core disease mutations occur interact to form an infra or intersubunit contact site close to the FKBP12 binding site and that mutations in either interacting partner alter both modulator binding and intersubunit communication. The specific aims are to: 1) delineate differences between the channel in the open and closed conformations to identify structural steps in channel gating, 2) assess the contributions of modulators to RYR1 structure, 3) localize the MH/CCD mutation sites and cysteine 3635 in the 3D structure of RYR1 and confirm protein-protein interactions. Structural information will be obtained using cryoelectron microscopy and computer reconstruction. Structural studies will be paralleled with functional analysis using single channel analysis of channels reconstituted into planar lipid bilayers and analysis of [3H]ryanodine binding. Interacting domains within RYR1 will be identified by assessing the ability of expressed fragments to interact using a biosensor assay and by identifying interacting regions using the yeast interaction trap.