SCREENING SCORPION, SPIDER, SNAKE, SNAIL TOXINS FOR BINDING TO K+ CHANNELS

筛选蝎子、蜘蛛、蛇、蜗牛毒素与 K 通道的结合

• 批准号: 7954047
• 负责人: RODERICK MACKINNON
• 金额: $ 4.98万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954047
• 关键词: Affinity Chromatography; Animals; Binding; Computer Retrieval of Information on Scientific Projects Database; Cysteine; Funding; Grant; Hydroxyproline; Institution; Libraries; Manuscripts; Mass Spectrum Analysis; Methodology; Methods; N-terminal; Potassium Channel; Proteins; Research; Research Personnel; Resources; Scorpions; Sequence Analysis; Snail Venoms; Snails; Snakes; Source; Spiders; Streptomyces lividans; Toxin; Tryptophan; United States National Institutes of Health; Venoms; Work; amidation; extracellular; inhibitor/antagonist; macromolecule; molecular mass; programs; rapid technique; tool; voltage gated channel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The extracellular entryway of the bacterial potassium channel of Streptomyces lividans (KcsA) is homologous to eukaryotic voltage gated channels. For this reason, KcsA is used as a template for the binding of extracellular pore blockers. Animal venoms such as snake, spider, scorpion and snail venoms are de facto libraries of naturally occurring toxins. Varrious venoms were screened against immobilized K+ channels using affinity chromatography. Following extensive washes, the channels were eluted along with specifically bound toxins. Mass spectrometry was used as a tool to quickly identify small protein toxins from their molecular mass and fragmentation spectra. This approach provides a rapid method for identifying potential inhibitors of eukaryotic potassium channels. We are developing mass spectrometric methods for rapidly determining the primary sequences of newly discovered channel-binding toxins. In particular, we have stablished methodology and workflow for toxin sequencing, including determination of number of cysteines and have developied a derivatization strategy to facilitate sequence analysis by ETD. Several toxins known and previously unknown have been identified. Several of the previously unknown toxins have sequenced mRNAs. PTMs like hydroxyproline, N-terminal amidation, and bromination of tryptophan were identified. We have also wriiten a program called TOXFINDER, which facilitates this analysis. We have prepared a manuscript for submission describing this work and plan to submit it shortly.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 Lividans链霉菌（KCSA）的细菌钾通道的细胞外入口与真核电压封闭通道同源。因此，KCSA用作结合细胞外孔隙阻滞剂的模板。蛇，蜘蛛，蝎子和蜗牛毒液等动物毒液实际上是天然存在的毒素的库。使用亲和色谱法对固定的K+通道进行筛选。大量洗涤后，将通道与特定结合的毒素一起洗脱。质谱法被用作快速从其分子质量和碎片光谱中识别小蛋白质毒素的工具。这种方法提供了一种快速的方法来鉴定真核钾通道的潜在抑制剂。我们正在开发质谱法，以快速确定新发现的通道结合毒素的主要序列。特别是，我们已经稳定了用于毒素测序的方法和工作流程，包括确定半胱氨酸的数量，并制定了一种衍生化策略，以促进ETD的序列分析。 已经确定了几种已知和以前未知的毒素。 几种先前未知的毒素已经测序mRNA。 鉴定了羟化，N末端肿瘤和色氨酸溴化的PTM。 我们还为一个名为Toxfinder的程序制定了一个计划，该程序促进了这一分析。 我们已经准备了一个手稿，用于提交描述这项工作，并计划尽快提交。