Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.
Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.
复制标题
心脏 Na 通道中 Zn2 和石房蛤毒素之间的竞争性结合相互作用。
DOI：
10.1016/s0006-3495(91)82269-x
复制
发表时间：
1991
影响因子：
3.4
通讯作者：
Moczydlowski,E
中科院分区：
文献类型：
--
作者：
Schild,L;Moczydlowski,E
哺乳动物心脏Na+通道对外源性锌离子阻断的亲和力比其他Na+通道亚型高约100倍。对于巴曲霉毒素修饰的狗或小牛心脏的Na+通道，外部锌离子的微摩尔浓度会导致闪烁阻塞到亚态水平，在-50 mV时电导约为开放通道的12%。我们通过对锌离子与钠离子通道相互作用的单通道分析，以及在巴曲霉毒素存在的情况下对平面脂质双层膜中单个心肌钠离子通道的化学修饰实验，验证了在这种阻断模式下，锌离子与心脏钠离子通道的一个亚位结合的假设。我们发现，外部的锌离子以严格竞争的方式解除了STX的阻挡作用。对这一现象的动力学分析与锌离子与STX在一个位置上直接结合竞争的方案一致，其本征平衡解离常数分别为30 nm和30微米。由于高亲和力的锌离子结合部位往往含有巯基作为该金属离子的配位配体，因此我们测试了一种硫基专一性的烷基化试剂--碘乙酰胺(IAA)对锌离子和STX阻断的影响。对于六个小牛心脏Na+通道，我们观察到暴露于5 mM IAA完全取消了锌离子阻断，并伴随着改变了STX结合，亲和力至少降低了20倍。这些结果导致我们提出了一个模型，在这个模型中，锌离子与心脏钠离子通道STX结合位点内或附近的一个亚位点结合。这个部位也被推定含有一个或多个半胱氨酸巯基。
Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.