Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter

A new method based on the toxicity of low intracellular pH (pH/sub i/) was developed to isolate fibroblasts 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 in a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pH/sub i/ recovery phase. The inhibition was provoked either by adding 5-(N-methyl, N-propyl)amiloride (MPA, LD50= 0.3 μM) 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 μM MPA. In response to an acid-load, these variants display a much higher rate of pH/sub i/ recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter. Alternatively reducing Na+ concentration of the pH/sub i/ 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 V/sub max/ and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. In conclusion, the genetic approach presented here: (i) provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and (ii) demonstrates that the external Na+-and amiloride-binding sites are not identical, since they can be genetically altered independently of each other