STRUCTURAL ANALYSIS OF THE CA++ RELEASE CHANNEL

骨骼肌钙离子释放通道或兰尼定受体(RYR1)跨膜区的开孔受多种调节剂与其胞质结构域结合的调节。导致人类两种疾病--中枢性核心病(CCD)和恶性热病(MH)的突变也经常在细胞质领域被发现。这项应用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。这种应用的工作假设是，调节器与跨膜结构域中变构调节孔的门控的亚基下或亚单位间接触位点结合并进行调节。我们还认为，恶性高热和中央核心疾病突变发生的两个细胞质结构域相互作用，形成靠近FKBP12结合位点的亚基下或亚单位间接触位点，其中任何一个相互作用的伙伴的突变都会改变调节子结合和亚基间通讯。其具体目的是：1)描述开放构象和闭合构象中通道的差异，以确定通道门控中的结构步骤；2)评估调节器对RYR1结构的贡献；3)在RYR1的3D结构中定位MH/CD突变位点和半胱氨酸3635，并确认蛋白质-蛋白质的相互作用。结构信息将使用低温电子显微镜和计算机重建获得。结构研究将与功能分析并行，使用重构为平面脂质双层的通道的单通道分析和[~3H]ryanodine结合的分析。RYR1内的相互作用区域将通过使用生物传感器试验评估表达片段的相互作用能力和通过使用酵母相互作用陷阱识别相互作用区域来确定。

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.