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

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 published a paper describing this work (B.M. Ueberheide, D. Feny¿, P.F. Alewood, B.T. Chait "Rapid, sensitive analysis of peptide venom components" Proc Natl Acad Sci, 106 (2009) 6910-5).