MOLECULAR IDENTIFICATION OF THE DANTROLENE RECEPTOR

丹曲林是一种抑制细胞内钙/2+释放的药物，是恶性高热的唯一治疗方法，恶性高热是一种对挥发性麻醉药的遗传敏感性。该药物在神经兴奋性毒性和脓毒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。本申请建议使用丹曲林的放射性标记、药理活性、光亲和类似物来鉴定、纯化和分子表征骨骼肌中可能的丹曲林受体。这些实验的结果将作为未来实验的基础，从而了解丹曲林受体在调节健康和疾病中细胞内钙/2+释放方面的作用。由于[~3H]丹曲林在骨骼肌的肌浆网中结合最多，该组织将被用作可能的受体来源。我们提供了一种定制合成的丹曲烯的光亲和类似物[~3H]叠氮杂多烯的数据，通过SDS-PAGE、PVDF膜的电印迹和随后的荧光照相确定，它具体地标记了大约140 kDa的蛋白质。作为一种分子标记方法，我们建议用标准的膜蛋白纯化方法来纯化丹曲林受体，这些方法包括制备凝胶电泳法、电洗脱法、洗涤剂增溶法、离子交换法、疏水作用法和/或亲和层析技术。一旦纯化，受体将接受蛋白质微测序。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之前描述的任何其他蛋白质显示同源性。对纯化蛋白或测序后构建的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 blotting检测骨骼肌以外的组织中可能存在的丹曲林受体，因为已有报道丹曲林有效地抑制了多种细胞类型的Ca/2+释放。免疫细胞化学将用于确定该蛋白相对于其他参与钙调节的已知蛋白的亚细胞定位。这些实验的结果将有助于设计未来的实验，帮助阐明丹曲林受体在兴奋-收缩偶联中的作用，并最终在其他组织的钙/2+调节中发挥作用。

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.