Transgenic Tethered Spider Toxins

由神经元离子通道功能障碍引起的人类病症是疼痛、痛苦和疾病的主要来源。 经济困难。通过了解正常的体内代谢， 生理功能由大量的生物药理学上不同的离子通道提供，这些离子通道存在于所有细胞中。 动物我们的长期目标是开发一种普遍适用的转基因工具包， 关于特定离子通道亚型的生理功能的假设的测试， 特定的神经回路。我们的方法是基于"拴系毒素"技术， 其中来自有毒捕食者的肽离子通道阻断剂表达为连接到 到细胞膜的细胞外。应用这种方法的初步研究表明， 栓系蜘蛛毒素作为细胞自主离子通道阻断剂，具有预期的靶向选择性 当表达在转基因果蝇大脑的特定神经回路中时。 初步研究还表明，澳大利亚漏斗网蜘蛛的毒液 含有多种多样的未表征的肽毒素，预期靶向多种离子通道 亚型因此，所提出的目的是针对（1）鉴定具有高效力的新型蜘蛛毒素 和（2）确定每一种的离子通道靶的分子身份 已识别的毒素第一个目标将通过筛选表达的特定行为效应来实现。 转基因蜘蛛大脑中的行为控制回路中存在许多不同的拴系漏斗网蜘蛛毒素， 苍蝇第二个目的将通过使用体外和体内电生理的组合来实现。 鉴定在第一个目标中鉴定的每种毒素的分子靶标的方法。拟议研究 将使整个果蝇神经生物学社区开始测试有关作用的假设， 特定的离子通道亚型在特定的神经回路在完整的行为动物， 对传统方法难以抗拒。由于生物物理和生理的广泛保护， 果蝇和哺乳动物之间的神经元功能机制，我们确定的新毒素不仅是 在活体苍蝇神经生物学上有巨大的用途，但也可以作为药理学试剂，用于探测结构 以及哺乳动物离子通道在健康和疾病中的功能。

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.