Molecular Characterization of Dantrolene Binding Site

描述(由申请人提供)： 我们的长期目标是了解丹曲林与细胞内RyR/Ca释放通道相互作用的结构和功能基础。我们已经确定骨骼肌兰尼定受体(RyR1)上名为DP1(a.a.590-609)的序列结构域是丹曲林的靶标，该区域与导致恶性高热易感性的突变密切相关。丹曲林似乎与RyR1相互作用，而不是与心脏RyR2相互作用，尽管DP1序列相同。因此，丹曲烯与RyR2的弱相互作用可能反映了通道其余部分或相关大分子给予的构象限制。关于丹曲烯与RyR亚型相互作用的分子基础的关键问题是：在RyR亚型中，决定丹曲烯结合和药理活性的语境要求是什么，既有结构域相互作用，又有蛋白质-蛋白质相互作用？这一假设的具体假设是，RyR上的590-609残基决定了丹曲烯结合位点的主要部分，该位点的构象决定了RyR亚型与丹曲林的特异性相互作用，并在调节这一钙释放通道中发挥了重要作用。设计了两个特定的目的来检验这一假说：目的1，确定丹曲林与RyRI相互作用的结构和生化基础。将使用光活性丹曲烯同系物的光亲和标记结合质谱学，以及鉴定序列的突变分析和钙荧光测量中的功能分析。目的2，从药理学、免疫学和分子水平探讨丹曲林对RyR1和RyR2的不同作用。RyR1-RyR2嵌合体将在异源和同源细胞中表达，并通过钙成像和光谱分析来探讨它们对丹曲林的反应。所获得的信息将为深入了解丹曲林干扰细胞内钙释放的分子机制并为治疗钙敏感型肌肉疾病提供合理的药物设计。

DESCRIPTION (provided by applicant): Our long term goal is to understand the structural and functional bases of the interaction of dantrolene with the intracellular RyR/Ca release channel. We have identified a sequence domain called DP1 (a. a. 590-609) on the skeletal muscle ryanodine receptor (RyR1) as a target for dantrolene, a region tightly linked to mutations resulting in susceptibility to malignant hyperthermia. Dantrolene appears to interact with RyR1 rather than cardiac RyR2 despite an identical DP1 sequence. The weak interaction of dantrolene with RyR2, therefore, may reflect conformational constraints imparted by the rest of the channel or associated macromolecules. Key question regarding the molecular basis of dantrolene interaction with RyR isoforms are: What are the contextual requirements, both domain-domain and protein-protein interactions that determine dantrolene binding and pharmacologic activity in RyR isoforms? The specific hypothesis for this proposal is that residues 590-609 on RyRldefine the major portion of the dantrolene binding site, and that conformation of this site determines the specific interaction of an RyR isoform with dantrolene and plays important role in the regulation of this Ca release channel. Two specific aims are designed to test this hypothesis: Aim 1, to define the structural and biochemical basis of dantrolene interaction with RyRI. Photoaffinity labeling with photoactive dantrolene congeners combined with mass spectrometry, and mutational analysis of identified sequences and functional analysis in Ca fluorescence measurements will be used. Aim 2, to explore the differential effects of dantrolene on RyR1 and RyR2 using pharmacological, immunological and molecular approaches. RyR1-RyR2 chimeras will be expressed in heterologous and homologous cells and their responses to dantrolene probed by Ca imaging and spectroscopy. The information gained will provide insight into the molecular mechanism by which dantrolene interferes with intracellular Ca release and lead to rational drug design for the therapy of Ca sensitive muscle diseases.