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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 变铅青链霉菌（Streptomyceslividans，KcsA）细菌钾通道的胞外通道与真核细胞的电压门控通道同源。因此，KcsA被用作细胞外孔阻断剂结合的模板。动物毒液，如蛇、蜘蛛、蝎子和蜗牛毒液，实际上是天然存在的毒素库。利用亲和色谱法对固定化K+通道筛选各种毒液。充分洗涤后，通道与特异性结合的毒素一起沿着洗脱。质谱法被用来作为一种工具，以快速识别小蛋白毒素从他们的分子质量和碎片光谱。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主要序列的质谱方法。特别是，我们已经建立了毒素测序的方法和工作流程，包括半胱氨酸数量的测定，并开发了衍生化策略，以促进ETD的序列分析。 一些已知的和以前未知的毒素已经被确定。 一些以前未知的毒素已经测序了mRNA。 鉴定了PTM，如羟脯氨酸、N-末端酰胺化和色氨酸溴化。 我们还编写了一个名为TOXFINDER的程序，它有助于这种分析。 我们已经准备了一份介绍这项工作的手稿，并计划很快提交。

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.