Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.
Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.
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化学选择性狼蛛毒素报告活细胞中野生型离子通道的电压激活。
DOI：
10.1073/pnas.1406876111
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发表时间：
2014
影响因子：
11.1
通讯作者：
Sack,JonT
中科院分区：
文献类型：
--
作者：
Tilley,DrewC;Eum,KennethS;Fletcher-Taylor,Sebastian;Austin,DanielC;Dupré,Christophe;Patrón,LilianA;Garcia,RitaL;Lam,Kit;Yarov-Yarovoy,Vladimir;Cohen,BruceE;Sack,JonT
电可兴奋细胞，如神经元，在其放电模式中表现出巨大的多样性，这是任何特定细胞中存在的离子通道复杂集合的结果。尽管有许多方法能够测量细胞电信号，但了解哪种类型的离子通道产生这些信号仍然是一个重大挑战。在这里，我们描述了使用一种新的机制来报告电压门控通道活性的外源性探针。我们合成了狼蛛毒素guangxitoxin-1 E（GxTX）的化学选择性衍生物，GxTX是一种抑制性胱氨酸结肽，可选择性地与Kv 2型电压门控钾通道结合。我们发现Kv2.1通道的电压激活触发GxTX解离，因此GxTX结合动态标记Kv 2激活。我们确定GxTX残基，可以取代巯基或炔轴承氨基酸，而不破坏毒素折叠或活性，并化学选择性连接荧光团或亲和探针到这些网站。我们发现，GxTX-荧光团共轭物与Kv2.1簇在活细胞中共定位，并从电压刺激激活的通道释放。Kv2.1激活可以用对细胞电流具有微小影响的探针浓度检测。与树枝状聚合物珠子缀合的化学选择性GxTX突变体同样结合表达Kv2.1的活细胞，并且珠子通过通道激活而释放。这些构象变化的光学传感器是原型探针，可以指示离子通道何时有助于电信号。
Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.