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

Tobias Wacker; Juan J. Garcia-Celma; Philipp Lewe; Susana L. A. Andrade

doi:10.1073/pnas.1406409111

Edited by H. Ronald Kaback, University of California, Los Angeles, CA, and approved June 4, 2014 (received for review April 8, 2014)

Significance

We have detected and analyzed electrogenic transport of ammonium and methylammonium by members of the ammonium transport (Amt) family of membrane proteins using solid-supported membrane electrophysiology. Amt transport is pH-dependent and occurs at a rate of 30–300 ions per s per trimer, well in the range of other transport proteins. The study establishes, to our knowledge, the first in vitro assay system for Amt transport in a fully controlled setup and settles debate about whether Amt proteins function as passive ammonia channels or active ammonium transporters.

Abstract

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.

Footnotes

↵¹To whom correspondence should be addressed. E-mail: andrade{at}bio.chemie.uni-freiburg.de.

Author contributions: T.W., J.J.G.-C., and S.L.A.A. designed research; T.W. and P.L. performed research; J.J.G.-C. contributed new reagents/analytic tools; T.W., J.J.G.-C., and S.L.A.A. analyzed data; and T.W., J.J.G.-C., and S.L.A.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406409111/-/DCSupplemental.