MOLECULAR IDENTIFICATION OF THE DANTROLENE RECEPTOR

丹曲林是一种抑制细胞内Ca/2+释放的药物，是恶性高热的唯一治疗方法，对挥发性麻醉剂具有遗传敏感性。该药物在神经兴奋性毒性和败血症模型中也显示出治疗前景。然而，该化合物的分子作用机制尚不清楚。本申请建议使用放射标记，药理活性，光亲和的丹曲林类似物，从骨骼肌中鉴定，纯化和分子表征假定的丹曲林受体。这些实验的结果将作为未来实验的基础，从而了解丹trolene受体在调节健康和疾病中细胞内Ca/2+释放中的作用。由于[3H]丹曲林结合在骨骼肌肌浆网中最大，因此该组织将被用作假定受体的来源。我们提供的数据表明，[3H]叠氮丹trolene是一种定制合成的、光亲和的丹trolene类似物，通过SDS-PAGE、电印迹到PVDF膜和随后的荧光成像确定，它可以特异性地光标记约140 kDa的蛋白质。利用这种方法作为分子标记，我们建议用膜蛋白纯化的标准方法来纯化假定的丹trolene受体，包括制备凝胶电泳、电洗脱、洗涤剂增溶、离子交换、疏水相互作用和/或亲和层析技术。一旦纯化，受体将进行蛋白质微测序。序列数据将与蛋白质数据库中的已知序列进行比较，以确定该蛋白质是否与任何其他先前描述的蛋白质具有同源性。测序后构建的纯化蛋白或独特合成肽的多克隆抗体将通过标准技术生产。这些抗体将用于Western blots检测除骨骼肌以外的组织中假定的丹曲洛烯受体，因为有报道称丹曲洛烯在多种细胞类型中有效抑制Ca/2+释放。免疫细胞化学将用于表征该蛋白的亚细胞定位与其他已知的蛋白质参与钙调节。这些实验的结果将有助于设计未来的实验，这些实验将有助于阐明丹trolene受体在兴奋-收缩耦合中的作用，并最终在其他组织中调节Ca/2+。

Dantrolene, a drug which inhibits intracellular Ca/2+ release is the only therapy of malignant hyperthermia, a genetic sensitivity to volatile anesthetics. The drug also shows therapeutic promise in models of neural excitotoxicity and sepsis. However, the molecular mechanism of action of this compound is not known. This application proposes to use a radiolabeled, pharmacologically active, photoaffinity analog of dantrolene, to identify, purify and molecularly characterize the putative dantrolene receptor from skeletal muscle. The results of these experiments will serve as the basis for future experiments leading to an understanding of the role of the dantrolene receptor in regulating intracellular Ca/2+ release in health and disease. Since [3H]dantrolene binding is greatest in the sarcoplasmic reticulum of skeletal muscle, this tissue will be used as a source of putative receptor. We present data that [3H]azidodantrolene, a custom synthesized, photoaffinity analog of dantrolene, specifically photolabels a protein of approximately 140 kDa, as determined by SDS-PAGE, electroblotting to PVDF membranes, and subsequent fluorography. By using this as a method of molecular tagging, we propose to purify the putative dantrolene receptor by standard methods of membrane protein purification, which includes preparative gel electrophoresis, electroelution, detergent solubilization, ion-exchange, hydrophobic interaction and/or affinity chromatography techniques. Once purified, the receptor will be subjected to protein microsequencing. Sequence data will be compared with known sequences from protein databases to determine whether this protein shows homology to any other previously described protein. Polyclonal antibodies to the purified protein or unique synthetic peptides constructed after sequencing will be produced by standard techniques. These antibodies will be used in Western blots to detect the putative dantrolene receptor in tissues other than skeletal muscle since it has been reported that dantrolene is effective in inhibiting Ca/2+ release in a variety of cell types. Immunocytochemistry will be used to characterize the subcellular localization of this protein with respect to other known proteins involved in calcium regulation. The results from these experiments will help in designing future experiments that would help elucidate the role of the dantrolene receptor in excitation- contraction coupling and, ultimately, in Ca/2+ regulation in other tissues.