Abstract：
Na+/H+ antiporters facilitate the exchange of Na+ for H+ across the cytoplasmic membrane in prokaryotic and eukaryotic cells. These transporters are crucial to maintain the homeostasis of sodium ions, consequently pH, and volume of the cells. Therefore, sodium/proton antiporters are considered promising therapeutic targets in humans. The Na+/H+ antiporter in Escherichia coli (Ec-NhaA), a prototype of cation-proton antiporter (CPA) family, transports two protons and one sodium (or Li+) in opposite direction. Previous mutagenesis experiments on Ec-NhaA have proposed Asp164, Asp163, and Asp133 amino acids with the significant implication in functional and structural integrity and create site for ion-binding. However, the mechanism and the sites for the binding of the two protons remain unknown and controversial which could be critical for pH regulation. In this study, we have explored the role of Glu78 in the regulation of pH by Ec-NhaA. Although we have created various mutants, E78C has shown a considerable effect on the stoichiometry of NhaA and presented comparable phenotypes. The ITC experiment has shown the binding of ~ 5 protons in response to the transport of one lithium ion. The phenotype analysis on selective medium showed a significant expression compared to WT Ec-NhaA. This represents the importance of Glu78 in transporting the H+ across the membrane where a single mutation with Cys amino acid alters the number of H+ significantly maintaining the activity of the protein.
摘要：
Na+/H+反转运蛋白促进了在原核和真核细胞中Na+与H+的跨细胞质膜的交换。这些转运蛋白对于维持钠离子的稳态至关重要，因此，pH值，和细胞的体积。因此,钠/质子反转运蛋白被认为是人类有前途的治疗靶点。大肠杆菌（Ec-NhaA）中的Na/H反转运蛋白，阳离子质子反转运蛋白（CPA）家族的原型，以相反的方向输送两个质子和一个钠（或Li+）。先前对Ec-NhaA的诱变实验提出了Asp164，Asp163和Asp133氨基酸，在功能和结构完整性方面具有重要意义，并创建了离子结合位点。然而,两个质子结合的机制和位点仍然未知且存在争议，这对于pH调节可能至关重要。在这项研究中,我们已经探索了Glu78在Ec-NhaA调节pH中的作用。虽然我们创造了各种各样的突变体，E78C对NhaA的化学计量显示出相当大的影响,并呈现可比较的表型。ITC实验已经显示了响应于一个锂离子的传输的〜5个质子的结合。在选择性培养基上的表型分析显示与WTEc-NhaA相比显著表达。这代表了Glu78在将H+转运穿过膜中的重要性,其中具有Cys氨基酸的单个突变改变了H+的数量,显著维持了蛋白质的活性。
