Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 2010 Nov 19;285(47):36656-65. doi: 10.1074/jbc.M110.161471. Epub 2010 Sep 15.
The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H(+) in exchange for one extracellular Na(+). We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227-249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp(238), Pro(239), and Glu(247). Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe(230), Gly(231), Ala(236), Val(237), Ala(244), Val(245), and Glu(248) were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229-236 and 239-250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn(227), Ile(233), and Leu(243) lining the translocation pore.
钠/氢交换体 1 同种型是一种广泛表达的完整膜蛋白。它位于细胞的质膜上，通过将细胞内的 H+排出，与细胞外的一个 Na+交换，来调节哺乳动物细胞内的 pH 值。我们使用半胱氨酸扫描突变和高分辨率 NMR 技术，研究了跨膜片段（TM）VI（残基 227-249）的结构和功能方面。在无半胱氨酸 NHE1 蛋白的背景下，将 TM VI 的每个残基突变为半胱氨酸，并确定了那些残基中对水可溶性巯基反应性化合物（2-（三甲基铵）乙基）甲硫磺酸酯（MTSET）和（2-磺酸盐乙基）甲硫磺酸酯（MTSES）敏感的残基。当突变为半胱氨酸时，三个残基基本上没有活性：天冬氨酸（238）、脯氨酸（239）和谷氨酸（247）。在其余残基中，突变体 N227C、I233C 和 L243C 的蛋白强烈被 MTSET 抑制，而苯丙氨酸（230）、甘氨酸（231）、丙氨酸（236）、缬氨酸（237）、丙氨酸（244）、缬氨酸（245）和谷氨酸（248）则被 MTSET 部分抑制。MTSES 不影响突变体 NHE1 蛋白的活性。使用高分辨率 NMR 光谱在十二烷基磷酸胆碱胶束中确定了代表 TM VI 的肽的结构。TM VI 包含两个彼此成直角取向的螺旋区域（残基 229-236 和 239-250），围绕着一个中央未缠绕区域。该结构与大肠杆菌蛋白 NhaA 的 TM IV 相似。结果表明，NHE1 的 TM VI 是一个不连续的孔衬螺旋，天冬酰胺（227）、异亮氨酸（233）和亮氨酸（243）残基排列在易位孔中。
