Lau Y H, Caswell A H, Garcia M, Letellier L
J Gen Physiol. 1979 Sep;74(3):335-49. doi: 10.1085/jgp.74.3.335.
The affinity and number of binding sites of [3H]ouabain to isolated transverse (T) tubules were determined in the absence and presence of deoxycholate. In both conditions the KD was approximately 53 nM while deoxycholate increased the number of binding sites from 3.5 to 37 pmol/mg protein. We concluded that the ouabain binding sites were located primarily on the inside of the isolated vesicle and that the vesicles were impermeable to ouabain. ATP induced a highly active Na+ accumulation by the T tubules which increased Na+ in the T tubular lumen by almost 200 nmol/mg protein. The accumulation had an initial fast phase lasting 2-3 min and a subsequent slow phase which continued for at least 40 min. The rate of the initial fast phase indicated a turnover number of 20 Na+/s. The Na+ accumulation was prevented by monensin but was unaffected by valinomycin. Ouabain did not influence Na+ uptake, but digitoxin inhibited it. At low K+ the accumulation of Na+ was reduced 3.7-fold below the value at 50 mM K+. 86Rb, employed as a tracer to detect K+, showed a first phase of K+ release while Na+ was accumulated. After 2-3 min, K+ was reaccumulated while Na+ continued to increase in the lumen. T tubules accumulated Cl- on addition of ATP. This suggested that ATP initiated an exchange of Na+ for K+ followed by uptake of Na+ and K+ accompanied by Cl-.
在有无脱氧胆酸盐的情况下，测定了[³H]哇巴因与分离的横管（T管）的结合位点亲和力和数量。在这两种情况下，解离常数（KD）约为53 nM，而脱氧胆酸盐使结合位点数从3.5 pmol/mg蛋白质增加到37 pmol/mg蛋白质。我们得出结论，哇巴因结合位点主要位于分离囊泡的内部，且囊泡对哇巴因是不可渗透的。ATP诱导T管高度活跃地积累Na⁺，使T管腔中的Na⁺增加了近200 nmol/mg蛋白质。这种积累有一个持续2 - 3分钟的初始快速阶段和随后至少持续40分钟的缓慢阶段。初始快速阶段的速率表明周转数为20个Na⁺/秒。莫能菌素可阻止Na⁺的积累，但缬氨霉素对其无影响。哇巴因不影响Na⁺的摄取，但地高辛会抑制它。在低钾浓度下，Na⁺的积累比在50 mM K⁺时的值降低了3.7倍。用作检测K⁺的示踪剂的⁸⁶Rb在Na⁺积累时显示出K⁺释放的第一阶段。2 - 3分钟后，K⁺重新积累，而Na⁺在管腔中继续增加。加入ATP后，T管积累Cl⁻。这表明ATP引发了Na⁺与K⁺的交换，随后伴随着Cl⁻摄取Na⁺和K⁺。
