Université Sorbonne Paris Cité, Université Paris Diderot, Inserm, INTS, Unité Biologie Intégrée du Globule Rouge, Laboratoire d'Excellence GR-Ex, 75739 Paris Cedex 15, France.
Sci Rep. 2017 Apr 7;7:46170. doi: 10.1038/srep46170.
Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO and Cl exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl/HCO exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO and outward Cl gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO for stomatin-deficient patients. Similarly, kinetics of Cl efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.
阴离子交换蛋白 1（AE1）和 stomatin 是红细胞（RBC）膜的完整蛋白。红系和肾脏的 AE1 在 HCO3 和 Cl 交换中起主要作用。stomatins 下调许多通道和转运蛋白的活性。生化研究表明，红系 AE1 与 stomatin 相互作用。此外，我们之前报道过 stomatin 缺陷 RBCs 中 AE1 表达水平正常。在这里，使用停流方法评估 stomatin 调节 AE1 依赖性 Cl/HCO3 交换的能力。在表达重组 AE1 和 stomatin 的 HEK293 细胞中，与仅表达内源性 stomatin 的细胞相比，过表达 stomatin 的细胞中与 AE1 活性相关的通透性高 30%。来自 stomatin 缺陷 RBCs 和对照的幽灵在存在 pH 或氯离子敏感荧光探针的情况下重新密封，并接受内向 HCO3 和外向 Cl 梯度。从碱化速率常数推断，stomatain 缺陷患者的 HCO3 通透性降低了 47%。同样，通过探针去猝灭来研究 Cl 外排的动力学，发现患者的通透性显著降低了 42%。原位邻近连接分析证实了 AE1 与 stomatin 在 HEK 重组细胞和 RBCs 中的相互作用。在这里，我们表明 stomatin 通过直接的蛋白质-蛋白质相互作用调节 AE1 的转运活性。
