Transgenic Tethered Spider Toxins

描述（由申请人提供）：由神经元离子通道功能障碍引起的人类疾病是疼痛、痛苦和经济困难的主要来源。通过了解所有动物中存在的大量生物物理上不同的离子通道所提供的正常体内生理功能，可以极大地促进它们的改善。我们的长期目标是开发一种普遍适用的转基因工具包，该工具包将允许在完整行为动物的特定神经回路中测试有关特定离子通道亚型生理功能的假设。我们的方法是基于“拴毒素”技术，其中来自有毒捕食者的肽离子通道阻滞剂以融合蛋白的形式表达，结合在质膜的细胞外侧。应用该方法的初步研究表明，系留蜘蛛毒素在转基因黑腹果蝇的大脑特定神经元回路中表达时，具有细胞自主离子通道阻滞剂的功能，并具有预期的目标选择性。初步研究还表明，澳大利亚漏斗网蜘蛛的Atracinae家族的毒液含有大量的未知肽毒素，有望针对各种各样的离子通道亚型。因此，提出的目标是针对(1)鉴定具有高效抗神经元离子通道的新型蜘蛛毒素和(2)确定每种鉴定毒素的离子通道靶点的分子特性。第一个目标将通过筛选在转基因果蝇大脑的行为控制回路中表达许多不同的系绳漏斗网蜘蛛毒素的特定行为效应来实现。第二个目标将通过结合体外和体内电生理方法来确定第一个目标中确定的每种毒素的分子靶点来实现。这项拟议的研究将使整个果蝇神经生物学社区能够开始测试关于特定离子通道亚型在完整行为动物的特定神经回路中的作用的假设，这些动物对传统方法是难以接受的。由于果蝇和哺乳动物神经元功能的生物物理和生理机制存在广泛的差异，因此我们鉴定的新毒素不仅将在体内果蝇神经生物学中具有巨大的应用价值，而且还将作为探索哺乳动物健康和疾病中离子通道结构和功能的药理学试剂。

DESCRIPTION (provided by applicant): Human disorders caused by neuronal ion channel dysfunction are a major source of pain, suffering, and economic hardship. Their amelioration can be greatly facilitated by understanding the normal in vivo physiological functions served by the large number of biophysically distinct ion channels present in all animals. Our long-term goal is the development of a generally-applicable transgenic toolkit that will permit the testing of hypotheses concerning the physiological functions of particular ion channel subtypes in specific neural circuits in intact behaving animals. Our approach is based on the "tethered toxin" technology, wherein peptide ion channel blockers from venomous predators are expressed as fusion proteins tethered to the extracellular side of the plasma membrane. Preliminary studies applying this approach indicate that tethered spider toxins function as cell-autonomous ion channel blockers with their expected target selectivity when expressed in specific neuronal circuits in the brains of transgenic Drosophila melanogaster fruit flies. Preliminary studies also indicate that the venoms of Australian funnel-web spiders of the Atracinae family contain a vast diversity of uncharacterized peptide toxins expected to target a wide variety of ion channel subtypes. The proposed aims are thus directed at (1) identifying novel spider toxins with high potency against neuronal ion channels and (2) determining the molecular identities of the ion channel targets of each identified toxin. The first aim will be achieved by screening for specific behavioral effects of expressing numerous different tethered funnel-web spider toxins in behavioral control circuits in the brains of transgenic flies. The second aim will be achieved by using a combination of in vitro and in vivo electrophysiological approaches to identify the molecular target(s) of each toxin identified in the first aim. The proposed research will enable the entire Drosophila neurobiology community to begin to test hypotheses concerning the roles of particular ion channel subtypes in specific neural circuits in intact behaving animals that have been refractory to traditional approaches. Because of the extensive conservation of biophysical and physiological mechanisms of neuronal function between flies and mammals, the novel toxins we identify will not only be of tremendous use for in vivo fly neurobiology, but also as pharmacological reagents for probing the structure and function of mammalian ion channels in health and disease.