Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel.
Biochemistry. 2012 Nov 27;51(47):9560-9. doi: 10.1021/bi301030x. Epub 2012 Nov 13.
pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and their pH regulation. Functional studies of NhaA in the membrane have yielded valuable information regarding its functionality in situ at physiological pH. Here, we Cys-scanned the discontinuous transmembrane segment (TM) IV (helices IVp and IVc connected by an extended chain) of NhaA to explore its functionality at physiological pH. We then tested the accessibility of the Cys replacements to the positively charged SH reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and the negatively charged 2-sulfonatoethyl methanethiosulfonate (MTSES) in intact cells at pH 8.5 and 6.5 and in parallel tested their accessibility to MTSET in high-pressure membranes at both pH values. We found that the outer membrane of E. coli TA16 acts as a partially permeable barrier to MTSET. Overcoming this technical problem, we revealed that (a) Cys replacement of the most conserved residues of TM IV strongly increases the apparent K(m) of NhaA to both Na(+) and Li(+), (b) the cationic passage of NhaA at physiological pH is lined by the most conserved and functionally important residues of TM IV, and (c) a pH shift from 6.5 to 8.5 induces conformational changes in helix IVp and in the extended chain at physiological pH.
所有细胞的 pH 和 Na(+) 稳态都需要 Na(+)/H(+) 反向转运蛋白。大肠杆菌主要反向转运蛋白 NhaA 的晶体结构为反向转运蛋白机制及其 pH 调节提供了普遍的见解。在膜中对 NhaA 的功能研究为其在生理 pH 下的原位功能提供了有价值的信息。在这里，我们通过半胱氨酸扫描不连续的跨膜片段（TM）IV（由延伸链连接的 IVp 和 IVc 螺旋）来探索其在生理 pH 下的功能。然后，我们测试了 Cys 替换物在 pH 值为 8.5 和 6.5 的完整细胞中对带正电荷的 SH 试剂[2-(三甲基铵)乙基]甲硫基磺酸盐溴化物（MTSET）和带负电荷的 2-磺基乙基甲硫基磺酸盐（MTSES）的可及性，并在两个 pH 值下的高压膜中平行测试了它们对 MTSET 的可及性。我们发现大肠杆菌 TA16 的外膜对 MTSET 起到部分渗透屏障的作用。克服这个技术问题后，我们揭示了：（a）TM IV 最保守残基的 Cys 替换强烈增加了 NhaA 对 Na(+) 和 Li(+)的表观 K(m)；（b）生理 pH 下 NhaA 的阳离子通道由 TM IV 中最保守和功能最重要的残基排列；（c）pH 值从 6.5 到 8.5 的变化诱导了生理 pH 下 IVp 螺旋和延伸链的构象变化。
