STRUCTURAL ANALYSIS OF THE CA++ RELEASE CHANNEL

骨骼肌Ca2+释放通道或ryanodine受体（RYR1）的跨膜区域孔的开放是通过多种调节剂与其细胞质结构域的结合来调节的。产生中央核心病（CCD）和恶性高热症（MH）这两种人类疾病的突变也经常在细胞质结构域发现。本应用的总体目标是阐明细胞质调节剂结合或突变存在改变通道门控的分子机制。这种应用的工作假设是，调节剂结合并调节亚基间或亚基间的接触位点，这些接触位点在跨膜区域变构地调节孔的门控。我们还提出，恶性高热和中心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结合位点的下或亚基间接触位点，任何一个相互作用伙伴的突变都改变了调节剂结合和亚基间通信。具体目的是：1)描述通道在开放和封闭构象中的差异，以确定通道门控的结构步骤；2)评估调节剂对RYR1结构的贡献；3)定位RYR1三维结构中的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。结构信息将通过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，使用单通道分析重组成平面脂质双层的通道和分析[3H]ryanodine结合。RYR1内的相互作用域将通过使用生物传感器测定评估表达片段的相互作用能力和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.