Institute for Biochemistry, Albert-Ludwigs University Freiburg, 79104 Freiburg, Germany;
Department of Biochemistry, University of Zürich, 8057 Zürich, Switzerland;Institute of Complex Systems-Cellular Biophysics Research Center Jülich, 52428 Jülich, Germany; and.
Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9995-10000. doi: 10.1073/pnas.1406409111. Epub 2014 Jun 23.
Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.
铵转运蛋白（Amt）是一类广泛存在的跨膜整合蛋白家族，它们能够特异性地转运铵跨膜运输。在原核生物、古菌和植物中，Amts 被用作环境 NH4(+) 清除剂，用于吸收和同化氮。在真核生物同源物——Rh 蛋白中，NH4(+)/NH3 的转运则用于肾脏的酸碱平衡和 pH 值稳定，或者用于红细胞中 NH4(+)/NH3（最终还有 CO2）的解毒。目前已有晶体结构和变体蛋白的相关研究，但由于底物的明确鉴定和转运事件的监测存在固有挑战，这严重阻碍了该领域的进一步发展。在这里，我们报告了一种可靠的体外测定法，该方法可定量 Amt 蛋白的产电性。我们使用基于固载膜（SSM）的电生理学方法，研究了来自广古菌 Archaeoglobus fulgidus 的三种 Amt 同源物。Af-Amt1 和 Af-Amt3 是产电性的，并且对铵和甲铵阳离子具有很高的特异性。转运是 pH 值依赖的，在 pH 值约为 5.0 时急剧下降。尽管存在显著的序列同源性，但三种蛋白质之间的功能差异变得明显。SSM 电生理学为普遍存在的铵转运蛋白提供了一种长期以来一直需要的功能性测定方法。
