Yun C H, Little P J, Nath S K, Levine S A, Pouyssegur J, Tse C M, Donowitz M
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Biochem Biophys Res Commun. 1993 Jun 15;193(2):532-9. doi: 10.1006/bbrc.1993.1656.
A family of Na+/H+ exchanger isoforms (called NHE1, NHE2, and NHE3) which exhibits a wide range of amiloride sensitivity has recently been cloned and characterized. A part of the domain, which determines amiloride sensitivity in the epithelial Na+/H+ exchanger isoform, NHE2, was identified by site-directed mutagenesis and functional studies using cDNAs stably expressed in a fibroblast cell line. It has previously been reported that AR300, an amiloride resistant mutant of the ubiquitous Na+/H+ exchanger isoform, NHE1, is 30-fold more resistant to methylpropyl amiloride (MPA) compared to NHE1 and contains a single amino acid substitution of L167F in the fourth putative transmembrane helix, which corresponds to L143 in NHE2. Therefore, in the present study point mutational substitutions were introduced into the equivalent of this fourth transmembrane helix of rabbit NHE2 (including Y144F; L143F; L143F and Y144F) to mimic the corresponding amino acids in NHE1, NHE3 (another epithelial isoform) and AR300, respectively. NHE2/L143F (mimicking NHE3) increased the IC50 for amiloride by 5-fold and for ethylisopropyl amiloride (EIPA) by 20-fold. Similarly, NHE2/L143F and Y144F (mimicking AR300) increased the resistance to both amiloride and EIPA by 10-fold. On the other hand, NHE2/Y144F (mimicking NHE1) did not affect the sensitivity to amiloride or EIPA, and this mutant, like wild type NHE2, is partially resistant to EIPA. Thus, amino acid 143 of NHE2 is critical for, but is not the only amino acid responsible for, amiloride and EIPA inhibition of Na+/H+ exchange. That none of the mutations studied altered the Na+ affinity of these Na+/H+ exchangers further suggests that amiloride binding and Na+ transport sites are not identical.
最近已克隆并鉴定出一个钠/氢交换异构体家族（称为NHE1、NHE2和NHE3），其对氨氯地平的敏感性范围很广。通过定点诱变和使用稳定表达于成纤维细胞系中的cDNA进行功能研究，确定了上皮钠/氢交换异构体NHE2中决定氨氯地平敏感性的部分结构域。此前有报道称，普遍存在的钠/氢交换异构体NHE1的氨氯地平抗性突变体AR300，与NHE1相比，对甲基丙基氨氯地平（MPA）的抗性高30倍，并且在第四个假定的跨膜螺旋中含有单个氨基酸取代L167F，这与NHE2中的L143相对应。因此，在本研究中，将点突变取代引入兔NHE2的该第四个跨膜螺旋的等效位置（包括Y144F；L143F；L143F和Y144F），以分别模拟NHE1、NHE3（另一种上皮异构体）和AR300中的相应氨基酸。NHE2/L143F（模拟NHE3）使氨氯地平的IC50增加了5倍，使乙基异丙基氨氯地平（EIPA）的IC50增加了20倍。同样，NHE2/L143F和Y144F（模拟AR300）使对氨氯地平和EIPA的抗性增加了10倍。另一方面，NHE2/Y144F（模拟NHE1）不影响对氨氯地平或EIPA的敏感性，并且该突变体与野生型NHE2一样，对EIPA具有部分抗性。因此，NHE2的氨基酸143对氨氯地平和EIPA抑制钠/氢交换至关重要，但不是唯一负责的氨基酸。所研究的突变均未改变这些钠/氢交换体的钠亲和力，这进一步表明氨氯地平结合位点和钠转运位点并不相同。
