PROPERTIES OF TETRODOTOXIN BINDING COMPONENT IN PLASMA-MEMBRANES ISOLATED FROM ELECTROPHORUS-ELECTRICUS
PROPERTIES OF TETRODOTOXIN BINDING COMPONENT IN PLASMA-MEMBRANES ISOLATED FROM ELECTROPHORUS-ELECTRICUS
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DOI：
10.1021/bi00650a002
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
RAFTERY, MA
中科院分区：
文献类型：
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作者：
REED, JK;RAFTERY, MA
以氚标记的河豚毒素（TTX）作为一种特异性膜探针，对电鳗电板中可电兴奋的Na⁺通道的生化特性进行了研究。电板的膜碎片主要通过差速离心法分离，并根据质膜标志物乙酰胆碱受体、乙酰胆碱酯酶、（Na⁺ + K⁺）ATP酶以及[³H]TTX结合情况进行了鉴定。平衡结合研究表明，[³H]TTX结合到单一的非相互作用受体位点群体上，表观解离常数为6±1×10⁻⁹ M。毒素 - 膜复合物以0.012秒⁻¹的一级速率常数解离。对复合物形成的pH依赖性研究表明，需要一个pK为5.3的可离子化官能团，且已证明该基团为羧基。用四氟硼酸三甲氧鎓（一种羧基修饰试剂）处理膜，导致[³H]TTX结合不可逆丧失，低浓度的TTX或石房蛤毒素可阻止这种情况发生。这种减少是由于结合位点总数减少，而非毒素结合亲和力降低。各种单价碱金属和多原子阳离子对TTX受体位点的相对结合亲和力表明，该位点显示出的阳离子辨别特性与先前在完整神经纤维中可电兴奋的Na⁺通道所报道的相似。提出了该位点在Na⁺通道离子选择性中的可能作用。
The biochemical properties of the electrically excitable Na+ channels in the electroplaque of E. electricus were investigated using tritiated tetrodotoxin (TTX) as a specific membrane probe. Membrane fragments from the electroplaque were isolated essentially by differential centrifugation and characterized with respect to the plasma membrane markers acetylcholine receptors, acetylcholinesterase, (Na+ + K+)ATPase and [3H]TTX binding. Equilibrium binding studies showed that [3H]TTX bound to a single population of noninteracting receptor sites with an apparent dissociation constant of 6 .+-. 1 .times. 10-9 M. The toxin-membrane complex dissociated with a 1st-order rate constant of 0.012 sec-1. Studies on the pH dependence of complex formation demonstrated the requirement for an ionizable, functional group with a pK of 5.3, and this group has been shown to be a carboxyl. Treatment of the membranes with trimethyloxonium tetrafluoroborate, a carboxyl group modifying reagent, resulted in an irreversible loss in the binding of [3H]TTX, which could be prevented by low concentrations of TTX or saxitoxin. This decrease was due to a reduction in the total number of binding sites and not to a decrease in toxin binding affinities. The relative binding affinities of various monovalent alkali metal and polyatomic cations for the TTX-receptor site showed that this site displayed cation discrimination properties which were similar to those reported previously for the electrically excitable Na+ channel in intact nerve fibers. A possible role for this site in the ion selectivity of the Na+ channel is proposed.