Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins

14.06.2014

Wacker T, Garcia-Celma JJ, Lewe P, Andrade SL.

Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9995-10000

Proc Natl Acad Sci U S A.       online article

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 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 around 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.