Reed J K, Raftery M A
Biochemistry. 1976 Mar 9;15(5):944-53. doi: 10.1021/bi00650a002.
The biochemical properties of the electrically excitable sodium channels in the electroplaque of Electrophorus 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 X 10(-9) M. The toxin-membrane complex dissociated with a first-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 sodium channel in intact nerve fibers. A possible role for this site in the ion selectivity of the sodium channel is proposed.
用电标记的河豚毒素（TTX）作为特异性膜探针，研究了电鳗电器官中电兴奋性钠通道的生化特性。基本上通过差速离心法分离出电器官的膜碎片，并根据质膜标记物乙酰胆碱受体、乙酰胆碱酯酶、（Na⁺ + K⁺）ATP酶和[³H]TTX结合情况进行表征。平衡结合研究表明，[³H]TTX与单一群体的非相互作用受体位点结合，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的可电离官能团，并且已证明该基团为羧基。用羧基修饰试剂四氟硼酸三甲氧鎓处理膜，导致[³H]TTX结合不可逆丧失，低浓度的TTX或石房蛤毒素可防止这种情况发生。这种减少是由于结合位点总数减少，而不是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，该位点具有阳离子识别特性，与完整神经纤维中电兴奋性钠通道先前报道的特性相似。提出了该位点在钠通道离子选择性中的可能作用。
