Chemical derivatization and purification of peptide-toxins for probing ion channel complexes.
Chemical derivatization and purification of peptide-toxins for probing ion channel complexes.
复制标题
用于探测离子通道复合物的肽毒素的化学衍生和纯化。
DOI：
10.1007/978-1-62703-345-9_2
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Kobertz,WilliamR
中科院分区：
文献类型：
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作者：
Hua,Zhengmao;Kobertz,WilliamR
离子通道是由离子传导α-亚基和调节β-亚基组成的多蛋白复合物。为了检测、鉴定和定量功能性K+通道复合物中的调节β-亚基，我们化学衍生了肽毒素，这些肽毒素可以与通道复合物中策略性放置的半胱氨酸残基特异性反应。用亲水性和疏水性双马来酰亚胺和其他分子探针衍生出肽毒素——白肉毒素的两种蛋白质标记方法已经被开发出来。利用这些半胱氨酸反应性肽毒素，我们专门针对在异种卵母细胞和哺乳动物细胞中表达的KCNQ1-KCNE1 K+通道复合物。试剂的模块化设计应该允许这种方法应用于涉及电兴奋性的许多离子通道复合物以及盐和水的平衡。
Ion channels function as multi-protein complexes made up of ion-conducting α-subunits and regulatory β-subunits. To detect, identify, and quantitate the regulatory β-subunits in functioning K+channel complexes, we have chemically derivatized peptide-toxins that specifically react with strategically placed cysteine residues in the channel complex. Two protein labeling approaches have been developed to derivatize the peptide-toxin, charybdotoxin, with hydrophilic and hydrophobic bismaleimides, and other molecular probes. Using these cysteine-reactive peptide-toxins, we have specifically targeted KCNQ1-KCNE1 K+channel complexes expressed in bothXenopusoocytes and mammalian cells. The modular design of the reagents should permit this approach to be applied to the many ion channel complexes involved in electrical excitability as well as salt and water homoeostasis.