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

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.Ammonium transport (Amt) proteins are a class of trimeric, integral membrane proteins found throughout all domains of life. Despite moderate primary sequence homologies, distinct family members from bacteria, archaea, and eukarya (including humans) share conserved structural features and a high number of conserved amino acid residues that are considered functionally relevant (1–4). Although the involvement of all Amt proteins in transporting NH₄⁺/NH₃ across biological membranes is undisputed, their functional context is diverse. Prokaryotes and plants use Amt proteins to scavenge NH₄⁺/NH₃—a preferred nitrogen source for cell growth—from their environment, whereas mammals use Amt orthologs, the Rhesus proteins, for detoxification and ion homeostasis in erythrocytes and in the kidney and liver tissues (1, 5, 6).Three decades ago, Kleiner and coworkers suggested that Amt proteins are secondary active and electrogenic transporters for ammonium (7–9). Various groups have subsequently confirmed this finding by two-electrode voltage-clamp experiments with protein produced recombinantly from RNA injected into Xenopus laevis oocytes. Here, plant Amt and Rhesus proteins were the main object of study, but some mechanistic details remained unclear, in particular the distinction between electrogenic NH₄⁺ uniport (10–13), NH₃/H⁺ symport (11, 12), or electroneutral NH₄⁺/H⁺ antiport (14, 15). In contrast, bacterial Amt proteins were described as passive channels for the uncharged gas ammonia (NH₃) (16). The first crystal structure for an Amt family member, AmtB from Escherichia coli (17), was interpreted to support this hypothesis, and an ongoing controversy concerning the transported species has persisted in the field ever since. Several points have been raised to challenge the possibility of gas channeling, the most critical of which seems to be that at physiological pH the protonation equilibrium of NH₃—with a pK_a of 9.4—would be >99% on the side of charged NH₄⁺. This point implies that the import of neutral ammonia gas must be preceded by extracellular deprotonation and followed immediately by intracellular protonation. In summary, the import of NH₃ would thus result in a net NH₄⁺/H⁺ antiport. Such a mechanism would be electroneutral, but it would be secondary active in the presence of a proton motive force, resulting in a vectorial pumping of ammonium out of the cell—which is, of course, physiologically unreasonable. A second point is that biological membranes are themselves highly permeable for uncharged ammonia, with a permeability coefficient, P_d = 10⁻³ cm·s⁻¹, similar to that of water (18), such that a dedicated transport protein would hardly be required. Westerhoff and coworkers have argued that active Amt transport thus is imperative and that cells must be able to quickly block Amt transport upon intracellular accumulation of ammonium to avoid uncoupling of the proton gradient through back-diffusion of NH₃ (19). In prokaryotes and some plants, this blocking is the task of regulatory GlnK proteins belonging to the signal transducing P_II family that bind to corresponding ammonium transporters when their regulatory ligand 2-oxoglutarate, the primary metabolic acceptor for NH₄⁺ during nitrogen assimilation, is depleted (20).The high expectations to understand the mechanism of Amt transport from 3D structures have not been met to date. The available structures of E. coli AmtB (17, 21) and its complex with GlnK (22, 23) of A. fulgidus Amt-1 (24), Nitrosomonas europaea Rh50 (25, 26), and human RhCG (27) all show the same, inward-facing state of the protein. Such apparent structural rigidity would match the picture of a fast channel, whereas active transport is generally considered to involve conformational changes that expose a binding site for the cargo molecule(s) alternatingly to either side of the membrane (28). In addition, the difficulties to detect NH₄⁺/NH₃ and to assay Amt transport led to a lack of functional studies carried out in vitro on well-defined systems. An uptake assay with AmtB reconstituted in proteoliposomes was described to provide evidence for passive gas channeling (17), but the methodology was later contested (2). Assays based on the detection of radioactive methylammonium (MA) uptake were only carried out in whole cells of E. coli, and studies with voltage-clamp electrophysiology using Amt-1 reconstituted in planar lipid bilayers did not yield conclusive results (our work). A series of potentially important variants have been produced (29–39), but the lack of an adequate functional assay has precluded definite conclusions.The debate concerning the transport mechanism of Amt proteins has not been settled to date, necessitating a reliable functional in vitro assay. The finding that electrogenic transport was observed in X. laevis oocytes, but not in the far smaller membrane patch of a planar lipid bilayer setup, suggested that the transport rate of Amt proteins was possibly too low to lead to a detectable current response, unless a larger number of protein units were incorporated into the bilayer. We have therefore focused on a controlled method of in vitro electrophysiology that allows the simultaneous activation of >10⁸ protein units, the solid-supported membrane (SSM) electrophysiology (40). With this approach, pioneered by Fendler and coworkers, we were able to detect robust ion currents from isolated and reconstituted Amt proteins.