Franchi A, Cragoe E, Pouysségur J
J Biol Chem. 1986 Nov 5;261(31):14614-20.
A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N-propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other.
基于低细胞内pH值（pHi）毒性开发了一种新方法，用于分离过表达Na+/H+逆向转运活性的成纤维细胞变体。将中国仓鼠肺成纤维细胞（CCL39）在含有50 mM氯化铵的培养基中孵育60分钟。当在pHi恢复阶段的60分钟内Na+/H+逆向转运体被抑制时，去除细胞外NH4+会导致快速且致命的细胞内酸化。通过添加5-（N-甲基，N-丙基）氨氯吡咪（MPA，半数致死剂量LD50 = 0.3 microM）或降低细胞外[Na+]（LD50 = 25 mM）来引发抑制。在酸负荷选择过程中逐渐增加MPA浓度，导致分离出两种稳定变体：AR40和AR300，它们分别对40和300 microM MPA具有抗性。响应酸负荷时，由于Na+/H+逆向转运活性的过表达，这些变体显示出更高的pHi恢复速率。此外，AR40和AR300的Na+/H+逆向转运体发生了改变：在AR300细胞中，抑制Na+/H+交换的MPA的半最大抑制浓度（K0.5）从5×10^(-8) M 转变为1.5×10^(-6) M，米氏常数（Km）（Na+）降低了2倍，最大反应速度（Vmax）增加了4.5倍。或者，逐步降低pHi恢复盐溶液培养基中的Na+浓度，导致选择另一类变体（DD8和DD12），其特征同样是Na+/H+逆向转运体发生改变且表达水平增加。DD12中氨氯吡咪敏感的Na+内流速率增加了10倍，这是由于最大反应速度增加了4倍以及对外位点处Na+或Li+的亲和力增加了2.5倍。有趣的是，DD12中对氨氯吡咪类似物MPA和对外源H+的亲和力没有变化。总之，本文提出的遗传方法：提供了一种通用且特异的方法，用于选择Na+/H+逆向转运体表达水平增加和/或结构改变的变体，并证明细胞外Na+结合位点和氨氯吡咪结合位点并不相同，因为它们可以彼此独立地发生遗传改变。
