Genetic Analysis of Conotoxin Targets

描述（由申请人提供）：拟议研究的目标是为突触和神经功能的研究提供基于遗传学的药理学。掠夺性锥面蜗牛毒液中毒素的巨大多样性提供了一种天然的基于组合的药理学，可用于选择性地结合介导突触通信的大量多样的神经递质受体组的成员。我们建议使用秀丽隐杆线虫来快速净化新的毒素，这将是广泛的神经生物学家感兴趣的。我们有三个主要目标。第一个目标是从圆锥螺的毒液中鉴定和纯化肽毒素，这种毒素通过扰乱神经系统功能来破坏土壤线虫的行为。该策略的长期目标是使用Conus毒素作为特异性探针，从而鉴定有助于神经系统功能的新基因产物。第二个目标是鉴定和纯化阻断秀丽隐杆线虫肌肉和神经元中特定配体门控电流的肽毒素。这些毒素对于秀丽隐杆线虫的神经生物学家来说将是无价的，并且将允许对秀丽隐杆线虫的突触传递进行详细的机制研究，目前由于缺乏特定的药物来急性阻断特定类别的电流，这是不可能的。我们将使用遗传和电生理策略来确定纯化的圆锥虫毒素的作用部位和机制。我们还使用组织特异性启动子在转基因蠕虫中表达活性Conus毒素。Conus Im1毒素在神经肌肉连接处阻断了大约50%的ach门控电流。导致这种im1敏感电流的基因产物尚未被确定。使用一种新的遗传策略，我们将确定这部分突触胆碱能电流所需的基因产物。Conus肽固有的巨大多样性，特异性和有效的受体相互作用，以及这些肽可以在转基因生物体中以组织特异性方式表达的事实，可能在未来为设计基于遗传的神经疾病治疗方法提供框架。

DESCRIPTION (provided by applicant): The goal of the proposed research is to provide a genetic-based pharmacology for the study of synaptic and neural function. The tremendous diversity of toxins from the venoms of predatory Conus snail species provide a natural combinatorial-based pharmacology that can be used to selectively bind to the members of the large and diverse group of neurotransmitter receptors that mediate synaptic communication. We propose to use C. elegans to rapidly purify new toxins that will be of interest to the broad community of neurobiologists. We have three major aims. The first aim is to identify and purify peptide toxins from the venoms of Conus snails that disrupt the behavior of the soil nematode C. elegans by perturbing nervous system function. A long-term goal of this strategy is to use Conus toxins as specific probes that will permit the identification of new gene products that contribute to nervous system function. The second aim is to identify and purify peptide toxins that block specific ligand-gated currents in muscles and neurons of C. elegans. These toxins will be invaluable for C. elegans neurobiologists and will allow for detailed mechanistic studies of synaptic transmission in C. elegans that currently are not possible because of the lack of specific pharmacological agents to acutely block specific classes of currents. We will use genetic and electrophysiological strategies to determine the site and mechanism of action of the purified Conus toxins. We have also used tissue-specific promoters to express active Conus toxins in transgenic worms. The Conus Im1 toxin blocks approximately 50% of the ACh-gated current at the neuromuscular junction. The gene products that contribute to this Im1-sensitive current have not yet been identified. Using a new genetic strategy, we will identify gene products that are required for this portion of the synaptic cholinergic current. The tremendous diversity inherent in Conus peptides, the specific and potent receptor interactions, and the fact that these peptides can be expressed in transgenic organisms in a tissue specific manner, may in the future provide a framework for designing genetic-based therapies for neurological disorders.