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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 变铅青链霉菌(Streptomyces lividans，KCSA)细菌钾通道的胞外通道与真核细胞电压门控通道同源。因此，KCSA被用作结合胞外孔道阻滞剂的模板。动物毒液，如蛇、蜘蛛、蝎子和蜗牛的毒液，实际上是自然产生的毒素的库。用亲和层析法筛选抗固定化K+通道的毒液。经过广泛的洗涤，这些通道与特定结合的毒素一起被洗脱。质谱学被用作一种工具，可以根据小蛋白毒素的分子质量和裂解光谱来快速识别它们。该方法为鉴定真核细胞钾通道的潜在抑制剂提供了一种快速的方法。我们正在开发快速确定新发现的通道结合毒素的初级序列的质谱学方法。特别是，我们已经建立了毒素测序的方法和工作流程，包括测定半胱氨酸的数量，并开发了一种衍生化策略，以促进ETD的序列分析。已确定了几种已知和以前未知的毒素。几种以前未知的毒素已经对mRNA进行了测序。鉴定出了羟脯氨酸、N-端酰胺化和色氨酸溴化等PTMS。

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.