Abstract
The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.
Highlights
The transport of ammonium across cell membranes is a fundamental biological process in all domains of life
The architecture of NeRh50 is highly representative of Rh proteins (Gruswitz et al, 2010; Lupo et al, 2007) which have been repeatedly reported to serve as electroneutral NH3 or CO2 gas channels (Cherif-Zahar et al, 2007; Hub et al, 2010a; Li et al, 2007; Lupo et al, 2007; Weidinger et al, 2007)
The activity of purified NeRh50 reconstituted into liposomes was quantified using Solid-Supported Membrane Electrophysiology (SSME) (Bazzone et al, 2017) experiments, where we recorded a NH4+-selective current (Figure 1) with a decay rate that is strongly dependent on the lipid-to-protein ratio (LPR; Table 1, Figure 1— figure supplement 1)
Summary
The transport of ammonium across cell membranes is a fundamental biological process in all domains of life. Rh mutations are linked to pathologies that include inherited hemolytic anemia, stomatocytosis, and early-onset depressive disorder (Huang and Ye, 2010) Despite this key general and biomedical importance, so far, no consensus on the pathway and mechanism of biological ammonium transport has been reached. High-resolution structures available for several Amt, Mep and Rh proteins show a strongly hydrophobic pore leading towards the cytoplasm (Andrade et al, 2005; Gruswitz et al, 2010; Khademi et al, 2004; Lupo et al, 2007; van den Berg et al, 2016) This observation led to the conclusion that the species translocated through Amt/Mep/Rh proteins is neutral NH3.
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