Renaud J F, Kazazoglou T, Lombet A, Chicheportiche R, Jaimovich E, Romey G, Lazdunski M
J Biol Chem. 1983 Jul 25;258(14):8799-805.
The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.
在不同发育阶段的大鼠心脏膜以及培养细胞中，研究了河豚毒素（TTX）与氚标记乙二胺类河豚毒素（[³H]en-TTX）的相互作用特性。通过对心脏培养细胞进行电生理学研究和²²Na⁺通量测量表明，Na⁺通道的功能形式对TTX的亲和力较低（250 - 700 nM）。对新生大鼠心脏培养细胞进行的结合实验（生物测定和[³H]en-TTX结合）表明，存在对TTX的高亲和力和低亲和力结合位点，其解离常数（Kd）分别为1.6和135 nM。在刚出生后的心脏匀浆中，[³H]en-TTX结合显示不存在对TTX的高亲和力结合位点，但存在一个Kd为125 nM的低亲和力结合位点。动力学研究和竞争实验证实了这一结果。相反，对成年心室匀浆和广泛纯化的膜进行的结合研究表明，存在对TTX的高亲和力和低亲和力结合位点，其Kd值分别为1.5和170 nM。低亲和力结合位点的最大结合容量比高亲和力结合位点高45倍。高亲和力位点在胎儿期或出生时不存在，但在出生后5天其数量逐渐增加，在出生后约45天达到最高水平。相反，低亲和力结合位点的数量在出生至成年期基本不变。出生后2天的心脏心肌细胞单层在体内没有高亲和力TTX结合位点，但在体外培养时会产生对TTX的高亲和力和低亲和力结合位点。本文给出的结果首次直接证明了大鼠心脏质膜中存在两类TTX结合位点。
