## 2 Background and Knowledge Gap
### Novelty and Rationale  
Understanding how small nutrients cross the outer membrane of Gram-negative bacteria is essential towards understanding how these organisms survive in nutrient-scarce environments [30; 31; 32; 33]. While active ammonium transporters (e.g., AmtB) are well-understood, the extent to which ammonium passes through general porins passively has not been directly measured [5; 6; 7; 19; 29]. This study demonstrated the application of an electrophysiological approach [34; 35; 36; 37] to quantify the transport of ammonia through the outer membrane general diffusion porins Omp-Pst2 from _Providencia stuartii_ and OmpF from _Escherichia coli_. OmpF is a classic example of a general diffusion porin found in the Gram-negative bacterium _Escherichia coli_[38; 39; 40; 41; 42; 43; 44; 45]. It forms a trimeric \(\beta\)-barrel channel, and the narrowest part of the channel is lined with charged residues [26; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48]. This architecture allows solutes up to approximately 600 Da to pass primarily according to their size and charge [38; 39; 40; 41; 42; 43; 44; 45; 49]. Omp-Pst2, which is found in _Providencia stuartii_, also forms a trimeric \(\beta\)-barrel, but it has been shown to conduct ions more readily [24; 27; 28]. Experiments from the literature have shown that Omp-Pst2 single-channel conductance is higher than that of its close relative, Omp-Pst1, and that it strongly prefers positively charged ion cations [24; 27; 28; 50]. Additionally, Omp-Pst2 opens and closes (gates) at lower voltages than many other porins, suggesting a specialization related to its handling of ammonium under stress [24; 27; 28]. This study directly compares how differences in porin architecture, the arrangement of charged residues, and the overall electrostatic environment influence ammonium versus counterion flow by measuring flux across two distinct general diffusion porins, Omp-Pst2 and OmpF [24; 27; 28]. For example, as reported in the literature, the constriction zone of Omp-Pst2 is slightly wider and contains additional acidic residues that likely create a more favorable pathway for ammonium than the narrower, more tightly charged constriction of OmpF [24; 28; 50]. In this study, a membrane-based electrical assay, specifically, the zero-current reversal potential technique [35; 36; 51], was used to investigate ammonium transport through membrane channels. By establishing a bi-ionic concentration gradient across a lipid bilayer reconstituted with membrane porins, reversal potentials were recorded, as described in prior studies [23; 25; 34; 36; 51; 52; 53; 54; 55]. In parallel, the single-channel conductance of individual porins were measured under similar conditions (Table 1). From the measured reversal potentials, the relative permeability ratios of cations to anions were calculated. These values, in combination with single-channel conductance data and bulk conductance data, allow for the estimation of ammonium flux through the porins. To ensure robustness, reversal potential measurements were performed using two different ammonium salts, namely, ammonium chloride and ammonium sulfate (Table 2). Additionally, staircase electrophysiology was employed to assess conductance for each porin in the presence of these salts (Tables 1 and 2), with all measurements conducted independently. While similar electrophysiological techniques have previously been applied to study the transport of antibiotics--such as cephalospor and carbapenem--through porins, typically reporting flux rates on the order of 10\({}^{2}\)-10\({}^{3}\) molecules per second, these methods have not been directly applied to ammonium transport in general diffusion porins [23; 25; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57]. Past studies have also explored how porins like Omp-Pst1 and Omp-Pst2 interact with antibiotics, often noting alterations in conductance profiles [24; 27; 28; 50].