Dept of Biochemistry, University of Alberta, Canada.
Dept of Chemistry, University of Alberta, Canada.
Biochim Biophys Acta Biomembr. 2019 Jan;1861(1):191-200. doi: 10.1016/j.bbamem.2018.07.014. Epub 2018 Jul 31.
The mammalian Na/H exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431-443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.
哺乳动物钠离子/氢离子交换体 1（NHE1）是一种完整的膜蛋白，通过在交换一个细胞外钠离子的同时去除一个细胞内质子来调节细胞内 pH（pH）。它参与心脏肥大和缺血再灌注对心脏的损伤，并增加其活性是触发乳腺癌转移的一个因素。NHE1 有一个广泛的 500 个氨基酸的 N 端膜结构域，介导运输，并由 12 个跨膜片段通过细胞内和细胞外环连接。细胞内环被假设调节对 pH 的敏感性。在这项研究中，我们对细胞内环 5（IL5），特别是氨基酸 431-443 的结构和功能进行了表征。将十一个残基突变为丙氨酸会导致部分或几乎完全抑制转运；值得注意的是，残基 L432、T433、I436、N437、R440 和 K443 的突变表明这些残基在 NHE1 功能中具有关键作用，而与靶向或表达无关。在膜模拟十二烷基硫酸钠溶液中，IL5 肽的核磁共振（NMR）溶液谱表明，IL5 具有稳定的三维结构和大量的 alpha 螺旋特征。NMR 化学位移表明 K438 与 W434 接近。总的来说，我们的结果表明，IL5 是一个关键的细胞内环，具有形成 alpha 螺旋的倾向，并且这个细胞内环的许多残基对质子感应和离子转运都很关键。
