Department of Biochemistry, University Alberta, Edmonton, AB T6G 2H7, Canada.
Instituto de Bioquímica Vegetal y Fotosíntesis Consejo Superior de Investigaciones Científicas, Avda, Américo Vespucio 49, 41092 Sevilla, Spain.
Int J Mol Sci. 2021 Oct 21;22(21):11349. doi: 10.3390/ijms222111349.
The mammalian Na/H exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser and Ser, that were similar in location and context to two amino acids of the Na/H exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783-790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.
哺乳动物 Na+/H+ 交换体 1（NHE1）是一种普遍存在于人体中的质膜蛋白。它通过去除细胞内质子并交换细胞外钠离子来调节细胞内 pH 值。它由 500 个氨基酸的膜结构域和 315 个氨基酸的调节胞质尾组成。在这里，我们研究了调节尾上两个氨基酸 Ser 和 Ser 突变的影响，这两个氨基酸在位置和上下文上与 Na+/H+交换体 SOS1 的两个氨基酸相似。将这两个氨基酸突变为 Ala 或磷酸模拟 Glu 不会影响表面靶向，但会导致表达的蛋白质水平略有降低。磷酸模拟突变会降低 NHE1 蛋白的活性，其作用是由于 Vmax 活性降低。Ser 到 Glu 的突变也导致全长蛋白和 NHE1 胞质尾的表观分子量发生变化。该区域的构象变化表明胰蛋白酶敏感性不同。我们还发现，含有氨基酸 783-790 的肽在体外蛋白质相互作用实验中与 NHE1 尾的几个更接近的区域结合。这些结果首次对这两个氨基酸进行了表征，并表明它们对酶动力学和 NHE1 蛋白的结构有重要影响。
