Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin
Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin
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DOI：
10.1021/bi060509s
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发表时间：
2006-08-22
期刊：
影响因子：
2.9
通讯作者：
Garcia, Maria L.
中科院分区：
文献类型：
--
作者：
Felix, John P.;Liu, Jessica;Garcia, Maria L.
内向整流钾通道（Kir）在细胞生理中起着重要作用。尽管代表了最简单的四聚体钾通道结构，但该通道家族的药理学仍在很大程度上未开发。在这方面，特硫平（tertiapin，TPN），一种从蜂毒中分离的21个氨基酸的肽，已被报道通过与这些通道的M1-M2接头区结合而以高亲和力抑制Kir1.1和Kir3.1/3.4通道。肽-通道相互作用的特征已经被电生理学探索，并且这些研究已经确定了在不影响其生物活性的情况下改变肽的组成的方法。在本研究中，TPN衍生物，TPN-Y1/K12/Q13，已合成和放射性标记的高比活度与I-125。TPN-Y1/K12/Q13和单碘-TPN-Y1/K12/Q13（[I-127]TPN-Y1/K12/Q13）以高亲和力抑制HEK 293细胞中稳定表达的大鼠而非人Kir1.1通道。[I-125]TPN-Y1/K12/Q13以可饱和、时间依赖性和可逆的方式与表达大鼠Kir1.1的HEK 293细胞以及这些细胞衍生的膜结合，结合反应的药理学与肽与Kir1.1通道结合一致。使用嵌合通道的研究表明，大鼠和人Kir1.1通道之间TPN敏感性的差异是由于M1-M2接头区域内存在两个非保守残基。当这些结果综合在一起时，它们表明[I-125] TPN-Y1/K12/Q13代表了用于研究大鼠Kir1.1通道的第一个高比活度放射性配体，并表明其可用于鉴定其他Kir通道调节剂。
Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with I-125. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([I-127]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [I-125]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [I-125] TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.