Pouysségur J, Sardet C, Franchi A, L'Allemain G, Paris S
Proc Natl Acad Sci U S A. 1984 Aug;81(15):4833-7. doi: 10.1073/pnas.81.15.4833.
A H+-suicide technique based on the reversibility of Na+/H+ antiport was developed for the selection of mutants deficient in this membrane-bound activity. The strategy was to use the Na+/H+ antiporter as a H+-vector killing device. Chinese hamster lung fibroblasts (CCL39) were loaded with LiCl and incubated in Na+-, Li+-free choline Cl saline solution (pH 5.5). Under these conditions, intracellular pH dropped in 5 min from 7.1 to 4.8, leading to a rapid loss of cell viability (less than 0.1% survival after 30 min). Cytoplasmic acidification and cell death were prevented by treatment with 5-N,N-dimethylamiloride, a potent inhibitor of Na+/H+ antiport. Of the H+-suicide resistant clones that survived two cycles of selection, 90% were found deficient in Na+/H+ antiport activity. One class of mutants (PS10, PS12) fully resistant to the H+-suicide test, does not acidify the cell interior in response to an outward-directed Li+ gradient and has no detectable amiloride-sensitive Na+ influx measured either in Li+- or H+-loaded cells. Growth of these fibroblast clones lacking Na+/H+ antiport was found to be pH conditional in HCO3(-)-free medium. Whereas wild-type cells can grow over a wide range of external pHs (6.6-8.2), PS mutants cannot grow at neutral and acidic pHs (pH less than 7.2); their optimal growth occurs at alkaline pH values (pH 8-8.3). These findings strongly suggest that the Na+/H+ antiport activity through regulation of intracellular pH plays a crucial role in growth control.
基于钠氢逆向转运体可逆性开发了一种氢离子自杀技术，用于筛选该膜结合活性缺陷的突变体。该策略是将钠氢逆向转运体用作氢离子载体杀伤装置。将氯化锂加载到中国仓鼠肺成纤维细胞（CCL39）中，并在无钠、无锂的氯化胆碱盐溶液（pH 5.5）中孵育。在这些条件下，细胞内pH在5分钟内从7.1降至4.8，导致细胞活力迅速丧失（30分钟后存活率低于0.1%）。用5-N，N-二甲基阿米洛利（一种有效的钠氢逆向转运抑制剂）处理可防止细胞质酸化和细胞死亡。在经历两轮筛选后存活的抗氢离子自杀克隆中，90%被发现缺乏钠氢逆向转运活性。一类对氢离子自杀试验完全抗性的突变体（PS10、PS12），在向外的锂离子梯度作用下不会使细胞内部酸化，并且在加载锂或氢离子的细胞中均未检测到阿米洛利敏感的钠内流。发现在无碳酸氢根的培养基中，这些缺乏钠氢逆向转运体的成纤维细胞克隆的生长具有pH依赖性。野生型细胞可以在很宽的外部pH范围内（6.6 - 8.2）生长，而PS突变体在中性和酸性pH（pH小于7.2）下不能生长；它们的最佳生长发生在碱性pH值（pH 8 - 8.3）。这些发现强烈表明，通过调节细胞内pH的钠氢逆向转运活性在生长控制中起关键作用。
