IDENTIFYING TOXINS THAT INTERACT WITH VOLTAGE GATED POTASSIUM CHANNELS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这个项目的目的是发现并测序新的毒素，这些毒素可以改变电压门控钾通道的门控。为了实现这一目标，在一个双层膜中筛选了几种(超过10种)不同狼蛛的粗毒，以对抗Pernix Aeropyrum Pernix的电压门控钾通道。智利斑马蜘蛛(Acanthogonatus Pissi)的粗毒表现出了非典型的结局。这里，流经通道的离子电流在接触毒液后增加，而不是更典型的电流减少的观察。粗毒经脱机高效液相色谱分离后，再对其进行双层筛选。发现了一小部分与粗毒的行为相似的东西。我们确定了有和没有感兴趣的毒液部分的通道的开放概率，很明显，开放概率随着毒素的存在而增加，并且当前的增加不是伪影。随后，对活性部分的两个主要成分进行了从头测序。这两种毒素含有6个半胱氨酸，半胱氨酸间距与已知狼蛛毒素相似，但半胱氨酸残基周围的残基与已知狼蛛毒素有很大不同。合成的多肽和质谱学证实了从头开始的序列。我们现在正在试图将这两种毒素折叠成它们的天然状态，并重复双层实验，以测试测序的毒素是否导致通道开放概率的增加。目前正在研究几种不同的折叠条件，这些条件都会导致不同的高效液相保留图谱。

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 aim of this project is to find and sequence new toxins that modify the gating of voltage gated potassium channels. Towards this goal, several (over 10) crude venoms of different tarantulas were screened in a bilayer set up against the voltage gated potassium channel from Aeropyrum pernix. The crude venom from Acanthogonatus pissi (Chilean zebra spider) showed an atypical outcome. Here the ion current through the channel increases upon exposure to venom as opposed to the more typical observation of current decrease. The crude venom was separated on offline HPLC and the bilayer screening repeated on the HPLC fractions. A fraction was found that mirrors the behaviors of the crude venom. We determined the open probability of the channel with and without the venom fraction of interest and it is clear that the open probability is increased with toxin present and that the current increase is not an artifact. Subsequently the two major components of the active fraction were de novo sequenced. These two toxins contain 6 cysteines and exhibit cysteine spacing similar to known tarantula toxins, but the residues surrounding the cysteine residues is quite different form known tarantula toxins. The de novo sequences were confirmed with synthetic peptides and mass spectrometry. Weare now in the process of trying to fold the two toxins into their native state and repeat the bilayer experiments to test whether the sequenced toxins cause the increase in the channel open probability. Several different folding conditions are currently under investigation that all lead to different HPLC retention profiles.