Mapping Ammonium Flux Across Bacterial Porins: A Novel Electrophysiological Assay with Antimicrobial Relevance

Abstract

This study presents a quantitative electrophysiological method to directly measure the passive transport of ammonium ions through bacterial outer membrane porins. Using a zero-current reversal potential assay in planar lipid bilayers under defined bi-ionic gradients, this study evaluates the permeability of ammonium salts through two general diffusion porins: Omp-Pst2 from Providencia stuartii and OmpF from Escherichia coli. Under matched ionic conditions, Omp-Pst2 exhibited significantly higher ammonium flux—approximately 6000 ions per second per monomer at a 1 µM gradient—compared to ~4000 ions per second for OmpF. Importantly, the identity of the accompanying anion (chloride vs. sulfate) modulated both the ion selectivity and flux rate, highlighting the influence of counterion interactions on porin-mediated transport. These findings underscore how structural differences between porins—such as pore geometry and charge distribution—govern ion permeability. The method applied here provides a robust framework for quantifying nutrient flux at the single-channel level and offers novel insights into how Gram-negative bacteria may adapt their membrane transport mechanisms under nitrogen-limited conditions. This work not only enhances our understanding of outer membrane permeability to small ions like ammonium, but also has implications for antimicrobial strategy development and biotechnological applications in nitrogen assimilation.

1. Introduction

Ammonium (NH₄⁺) is a primary nitrogen source for bacteria and is essential for processes such as amino acid and nucleotide synthesis and energy metabolism [1,2]. In Gram-negative pathogens such as Providencia stuartii, which frequently cause catheter-associated urinary tract infections, efficient ammonium acquisition can determine survival and virulence under nutrient-limited conditions [3,4]. While active transporters (e.g., AmtB family proteins) have been well-characterized, the role of passive diffusion through outer membrane porins is not well-understood [3,4,5,6,7]. Current methods, such as growth-based assays or radiotracer measurements, only provide indirect estimates of total ammonium uptake and lack single-channel resolution [8,9]. Consequently, fundamental questions persist regarding how porins modulate passive ammonium flux, especially at the low micromolar gradients encountered in vivo [10,11,12].

2. Background and Knowledge Gap

Gram-negative bacteria have a multilayered cell envelope that consists of an inner (cytoplasmic) membrane, a periplasmic peptidoglycan layer, and an outer membrane [13,14,15,16,17,18]. The outer membrane functions as both a selective barrier against harmful agents and a gateway for small hydrophilic solutes, including ammonium [7,16,17,18,19,20]. Trimeric β-barrel proteins, known as porins/membrane proteins, form aqueous channels that allow ions and small molecules to pass passively along electrochemical gradients [16,17,18,21,22,23]. General diffusion porins, such as OmpF in Escherichia coli and Omp-Pst2 in Providencia stuartii, provide pathways whose throughput depends on pore architecture and ion concentrations on either side of the membrane [16,22,23,24,25,26,27,28].

To date, understanding of ammonium movement through porins has primarily been based on indirect observations. Measurements such as total nitrogen consumption or per-cell ammonium uptake may reflect the combined influence of multiple transport mechanisms. While high-resolution structural studies—for example, the 1.35 Å structure of AmtB—have provided insights into active ammonium transport, comparable structural or electrophysiological data specifically related to porin-mediated ammonium flux appear to be limited [5,6,7,19,29].

2.1. 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 β-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 β-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 [23,25,34,35,36,51,52,53,54,55]. In parallel, the single-channel conductance of individual porins were measured under similar conditions (

Table 1. Bi-ionic permeability ratios. Calculated cation flux rate of the different substrates $\left(P_{{anion}^{-}}/P_{{\mathit{c}\mathit{a}\mathit{t}\mathit{i}\mathit{o}\mathit{n}}^{+}}\right)$ for the current flux through Omp-Pst2 and OmpF in ammonium sulfate and ammonium chloride, respectively. Bi-ionic reversal potential $V_{rev}$ needed to obtain zero current for a concentration gradient for the respective substrate only. Using the Goldmann–Hodgkin–Katz equation gives the permeability ratio $P_{anion-}/P_{cation+}$ for the ion current flux through Omp-Pst2 and OmpF [23].

	Substrate	Gradient (Cis vs. Trans)	Vrev (mV) (Experiment)	${\mathit{P}}_{{\mathit{a}\mathit{n}\mathit{i}\mathit{o}\mathit{n}}^{-}}/{\mathit{P}}_{{\mathit{c}\mathit{a}\mathit{t}\mathit{i}\mathit{o}\mathit{n}}^{+}}$
Omp-Pst2	Ammonium chloride	200/50 mM	22.46 ± 7	1:05
	Ammonium sulfate	200/50 mM	20.06 ± 5	1:06
OmpF	Ammonium chloride	200/50 mM	18.5 ± 5	1:09
	Ammonium sulfate	200/50 mM	18±4	1:09

).

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. Estimated ammonium ion flux through Omp-Pst2 and OmpF porins under defined bi-ionic gradients.

Porin	Salt	G (Trimer) [nS]	G (Mono) [nS]	Vrev [mV]	Bulk Cond. [mS/cm] 50mM Salt	Calculated Flux @ 1 µM (ions/s)
Omp-Pst2	NH4Cl	0.53 ± 0.29	0.177	22.46	6.2	≈6680
Omp-Pst2	(NH4)2SO4	1.17 ± 0.6	0.39	20.06	11.78	≈5520
OmpF	NH4Cl	0.4 ± 0.25	0.133	18.5	6.2	≈4300
OmpF	(NH4)2SO4	1.1 ± 0.6	0.367	18	11.78	≈5150

). Additionally, staircase electrophysiology was employed to assess conductance for each porin in the presence of these salts (Table 1 and Table 2), with all measurements conducted independently.

While similar electrophysiological techniques have previously been applied to study the transport of antibiotics—such as cephalosporins and carbapenems—through porins, typically reporting flux rates on the order of 10²–10³ molecules per second, these methods have not been directly applied to ammonium transport in general diffusion porins [23,25,35,36,37,51,52,53,54,55,56,57]. 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].

In contrast, the current work focuses on ammonium, a small cations (~18 Da) that does not induce transient blockage events commonly seen with larger substrates. Instead, by leveraging reversal potential-based assays, this study offers direct electrophysiological measurements of ammonium flux through general diffusion porins—providing new insights into a transport mechanism.

2.2. Physiological Relevance Under Low-Ammonium Conditions

In many natural and host environments, such as the urinary tract or certain soil microenvironments, external ammonium concentrations can drop to the low micromolar range (10 µM or less) [58,59,60]. Under such conditions, passive diffusion through porins may become the main route for ammonium to reach the periplasm [16,18,21,61]. When active transporters are either saturated or regulated down, the permeability of the porins can determine how quickly the cell acquires nitrogen. By modeling a realistic gradient of approximately 1 µM ammonium across the outer membrane, the measured flux rates demonstrate how quickly a bacterium can gather ammonium passively. The finding that Omp-Pst2 can move approximately 6,000 ammonium ions per second under such a small gradient suggests that porin-mediated uptake could account for a significant portion of Providencia stuartii’s total ammonium acquisition when nitrogen is limited [1,62,63].

From a therapeutic perspective, these findings point toward two possible antimicrobial strategies:

3. Porin Inhibition

When bacteria face nitrogen scarcity, such as during infection in environments with very low ammonium, blocking a high-throughput porin like Omp-Pst2 could dramatically reduce ammonium uptake. With less nitrogen available, bacterial growth and virulence would be impaired [5,64,65].

4. Porin-Mediated Prodrug Delivery

Designing antibiotic prodrugs that mimic ammonium or pair with anions preferred by Omp-Pst2 could enable more efficient entry into the periplasm through these porins [66,67]. Exploiting the porin’s natural preference for ammonium could allow such prodrugs to achieve higher local concentrations inside Gram-negative pathogens, potentially overcoming common resistance mechanisms like efflux pumps [68].

4.1. Hypothesis and Objectives

It is hypothesized that Omp-Pst2 facilitates a higher passive flux of ammonium than OmpF does, due to differences in pore geometry, charge distribution, and lining residues that favor the permeation of NH₄⁺. To test this hypothesis, purified Omp-Pst2 and OmpF were reconstituted into planar lipid bilayers under controlled experimental conditions [23,35,36]. Zero-current reversal potentials were measured across defined ammonium salt gradients, and permeability and flux values were calculated accordingly [25,35,36,51,54].

4.2. Significance and Scope

Nitrogen uptake is essential for bacterial growth and virulence, particularly in Gram-negative species, where outer membrane porins serve as primary gateways for small nutrients, such as ammonium [1,6,19,63,69,70]. While active transporters, such as AmtB, have been studied extensively, passive diffusion through general porins, like OmpF in Escherichia coli and Omp-Pst2 in Providencia stuartii, can become an important route when external ammonium levels are low [5,6,7,24,25,26,28,38,41,50,71,72]. Until now, measurements of ammonium uptake have typically used whole-cell assays or radiotracer methods, which cannot distinguish between active and passive pathways [5,6,7,19,63,69]. This study closes that gap by directly measuring, for the first time, how much ammonium passes through individual porin channels under controlled ionic conditions [35,36]. By comparing Omp-Pst2 and OmpF simultaneously, this study reveals how differences in pore size, the arrangement of charged residues, and the narrow “constriction zone” affect the rate and selectivity of ammonium diffusion. Generally, porins form water-filled β-barrel channels without specific binding sites; therefore, molecules below approximately 600 Da can pass based on their size and charge [36,48,49,72,73]. In this case, Omp-Pst2’s slightly wider channel and unique charge pattern appear may allow more ammonium flux than OmpF. However, detailed, atomic-level confirmation will require future structural or computational studies.

Under nutrient-limiting conditions, such as those found in host environments where ammonium concentrations drop into the micromolar range, porin-mediated diffusion may become a limiting factor in nitrogen acquisition [1,29,63]. Many Gram-negative pathogens respond to nitrogen scarcity by increasing porin expression. Thus, blocking a key porin, such as Omp-Pst2, could slow growth or prevent biofilm formation under these conditions, offering a novel antimicrobial strategy [74,75].

Conversely, identifying which porin channels favor ammonium influx suggests a route for drug delivery [16,17,18,21,61,76,77]. Designing antibiotic prodrugs that mimic ammonium or pair with counterions preferred by Omp-Pst2 or OmpF could drive higher drug concentrations into the periplasm of Gram-negative bacteria. This approach could bypass common resistance mechanisms, such as efflux pumps, and improve antibiotic effectiveness.

Finally, providing quantitative, single-channel measurements of ammonium flux lays the groundwork for more accurate models of how bacteria acquire nitrogen in complex environments. Incorporating these detailed porin data into computational simulations could improve predictions of growth under nutrient-limited conditions, competition among microbial communities, and the development of resistance under selective pressure [1,60,64,65,68]. Overall, this study provides a new tool for investigating porin function, enables the design of targeted antimicrobials, and closes a longstanding gap in our understanding of bacterial nitrogen uptake.

5. Materials and Methods

Ammonium chloride, and ammonium sulfate were kindly obtained from Sigma Aldrich (Schnelldorf, Germany), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine was purchased from Avanti Polar Lipids (Alabaster, AL, USA). All other chemicals used in this study were procured from AppliChem. Prof. Mathias Winterhalter, Bremen, Germany provided OmpF, Omp-pst2, and all the chemicals, as well as access to laboratory space, instruments, and all necessary resources for experimental measurements.

5.1. Planar Lipid Bilayer and Electrical Recording:

Planar lipid bilayer was formed as described in detail elsewhere [28,35,36]. An aperture in a Teflon septum with a diameter of 105-130 μm was pre-painted with hexadecane dissolved in purified n-hexane at 1.5–2% (v/v) and the Teflon chambers were dried for 40–45 min. Bilayers were made with 1,2-diphytanoyl-sn-glycerophosphocholine at a concentration of 5 mg/mL in n-pentane [25,35,36,51,54]. Stock solutions of the outer membrane porins Omp-Pst2/OmpF, were added to the cis side for all the measurements. Standard Ag/AgCl calomel electrodes (Metrohm AG) were employed to perceive the ionic current [35,36]. The cis side electrode of the cell was connected to the ground, whereas the trans side electrode was connected to the headstage of an Axopatch 700A amplifier, used for the conductance measurements in the voltage clamp mode. Signals were filtered by an on-board low-pass Bessel filter at 10 kHz and recorded onto a computer hard drive with a sampling frequency of 50 kHz [35,36]. The analysis of the current recordings was performed using Clampfit (Axon Instruments, Union City, CA, USA) and Origin^R (Origin-Lab-Corp, Northampton, MA, USA). The current voltage relation of the individual experiments was calculated from single averaged currents at the given voltage [35,36]. All the experiments were repeated three times minimum. Standard solutions along with bulk conductance and channel conductance are given in Table 2 [35,36,37]. The relative permeability of cations vs. anions in the bi-ionic case were obtained by fitting the experimental I-V-curves with the Goldman–Hodgkin–Katz current equation [35,36,78]. Single-channel conductance in the presence of ammonium chloride and ammonium sulfate was measured for Omp-Pst2/OmpF reconstituted in planar lipid bilayer.

5.2. Single Channel Recording Under Asymmetric Conditions: Bi-ionic Potential

To gain evidence on the permeability of Omp-Pst2 or OmpF for the ammonia cations, this work applied an experimental approach based on the Goldman–Hodgkin–Katz (GHK) current equation [35,36,37]. The selectivity of Omp-Pst2/OmpF in an artificial bilayer membrane under bi-ionic environments on both sites of the planar bilayer [35,36] was measured. Since ion fluxes are created by concentration gradients, the GHK current equation permits the calculation of the relative ion permeability by the macroscopic GHK theory, by means of the chemical potential created by the different electrophoretic mobility of the ions themselves [36,78,79]. Membrane proteins Omp-Pst2 and OmpF were first reconstituted in a 50 mM solution of ammonium sulfate and ammonium chloride in separate measurements; the ion concentration was then raised in one side (the cis side corresponding to the electrical ground side) to 200 mM. I/V (Figure 1) IV curves were recorded. As cations and anions have different permeability, a shift in the zero-current potential is observed, called reversal potential. Using the GHK equation allows us to calculate the permeability ratio $P_{cation}/P_{anion}$:

$$∆V=RT/F\times ln\left({\displaystyle \frac{P_{cation}\times {\left[cation\right]}^{cis}+P_{anion}\times {\left[anion\right]}^{trans}}{P_{cation}\times { \left[cation\right]}^{trans}+P_{anion}\times {\left[anion\right]}^{cis}}}\right)$$

(1)

where ∆V is the measured reversal potential, P_cat the permeability for cation⁺, and P the permeability for anion⁻ (the universal gas constant R = 8.3 J/mol⁻¹ K⁻¹, and the Faraday constant F = 9.6 10⁴ C/mol⁻¹) [23,34,35,36].

The permeability ratios for ammonium sulfate and ammonium chloride in Omp-Pst2 and OmpF under bionic conditions are reported in Table 1. For the estimation of single-channel permeability, the conductance of a bi-ionic salt solution gives the total flux of ions under a given external voltage and the permeability ratio allows the ion flux to be distributed on the respective cations and anions [23,34,35,36]. However, the concentration driven flux is less obvious as both charges move in the same direction and only the difference gives rise to an electrical signal [23,34,35,36]. The reversal potential balances both fluxes and can be used to estimate the strength [23,34,35,36]. Extrapolation to 1µM results in the molecular flux of molecules (molecules/s) (Table 2). Furthermore, the data reveal that both channels are capable of transporting ammonium ions, but they differ in their efficiency and selectivity [23,34,35,36]. For Omp-Pst2, the use of ammonium chloride results in a higher reversal potential and a moderate permeability ratio ≈ (1:5), correlating with a higher ion flux rate. In contrast, OmpF shows a much higher permeability ratio ≈ (1:9) when ammonium chloride or ammonium sulfate is used, indicating a stronger selectivity for ammonium ions but with a lower extrapolated flux rate. These detailed observations suggest that the structural differences between Omp-Pst2 and OmpF may contribute to their distinct transport properties under similar ionic conditions, with Omp-Pst2 generally supporting a higher rate of ammonium transport compared to OmpF.

5.3. Ammonium Sulfate (Figure 1)

Current recordings from bilayers containing multiple outer membrane porins OmpPst2 in the presence of symmetrical 50 mM ammonium sulfate show clearly detectable single pore insertion (Figure 1A) but with high channel noise. The analysis of the single pore currents [35] (Figure 1B) using current histograms for single pore gating [35] revealed that the bilayer contained ≈ 30 active trimeric OmpPst2 channels, while the single trimeric channel pore conductance in the presence of symmetrical 50 mM (NH₄)₂SO₄ was $G_{sp}=1.17 nS$ (Table 1).

Figure 1. (A). Current recordings from a bilayer in symmetrical 50 mM (cis/trans) (NH₄)₂SO₄ at V_m = 150 mV after the application of, typically, 2-3 µL with a protein concentration of ≈5 mg/mL at t = 0. After 100 s, when V_m = 150 mV was switched to V_m = 0, n ≈ 30 trimeric OmpPst2 channels had been incorporated into the bilayer. (B). Current amplitude histogram (from A).

Figure 1. (A). Current recordings from a bilayer in symmetrical 50 mM (cis/trans) (NH₄)₂SO₄ at V_m = 150 mV after the application of, typically, 2-3 µL with a protein concentration of ≈5 mg/mL at t = 0. After 100 s, when V_m = 150 mV was switched to V_m = 0, n ≈ 30 trimeric OmpPst2 channels had been incorporated into the bilayer. (B). Current amplitude histogram (from A).

5.4. Ammonium Chloride (Figure 2)

Current recordings from bilayers containing multiple outer membrane porins OmpPst2 in the presence of symmetrical 50 mM ammonium chloride show clearly detectable single pore insertion (Figure 2A) but with high channel noise. The analysis of the single pore currents (Figure 2B) using current histograms for single pore gating [35] revealed that the bilayer contained ≈100 active trimeric OmpPst2 channels, while the single trimeric channel pore conductance in the presence of symmetrical 50 mM (NH₄)Cl was ${\mathit{G}}_{\mathit{s}\mathit{p}}=\mathbf{0.53} \mathbf{n}\mathbf{S}$ (Table 1).

Figure 2. (A). Current recordings from a bilayer in symmetrical 50 mM (cis/trans) NH₄Cl at V_m = 150 mV after the application of, typically, 2–3 µL with a protein concentration of ≈3.2 mg/mL at t = 0. After 100 s, when V_m = 150 mV was switched to V_m = 0, n ≈ 100 trimeric OmpPst2 channels had been incorporated into the bilayer. (B). Current amplitude histogram (from A).

Figure 2. (A). Current recordings from a bilayer in symmetrical 50 mM (cis/trans) NH₄Cl at V_m = 150 mV after the application of, typically, 2–3 µL with a protein concentration of ≈3.2 mg/mL at t = 0. After 100 s, when V_m = 150 mV was switched to V_m = 0, n ≈ 100 trimeric OmpPst2 channels had been incorporated into the bilayer. (B). Current amplitude histogram (from A).

Each entry reports the recalculated flux of cations (in ions per second per monomer) for Omp-Pst2 and OmpF, based on single-channel conductance values (G), experimentally measured reversal potentials (Vrev), and bulk solution conductance under symmetric 50 mM salt conditions. Flux rates were extrapolated to a 1 µM transmembrane gradient using the Goldman–Hodgkin–Katz (GHK) framework and converted to molecular throughput assuming a constant elementary charge (1.6 × 10⁻¹⁹ °C). All salts were tested in independent planar lipid bilayer recordings under matched cis/trans gradients (200/50 mM). Results highlight the higher overall ammonium permeability of Omp-Pst2 relative to OmpF and the influence of counterion identity on transport efficiency.

6. Results and Discussion

To directly compare how two general diffusion porins—Omp-Pst2 from Providencia stuartii and OmpF from Escherichia coli—facilitate ammonium (NH₄⁺) passage, each porin was reconstituted into planar lipid bilayers and applied well-controlled bi-ionic gradients [35,36,53]. By measuring zero-current reversal potentials under defined salt conditions and fitting the resulting I–V relationships to the Goldman–Hodgkin–Katz equation, permeability ratios (P−/P+) and extrapolated single-channel flux rates [35,36,53] at physiologically relevant (micromolar) NH₄⁺ concentrations were obtained. This head-to-head approach reveals both quantitative transport efficiencies and selectivity differences that underlie ammonium uptake in bacterial outer membranes. When Omp-Pst2 was assayed with an ammonium chloride gradient (200 mM cis∕50 mM trans), the observed reversal potential (V_rev) was 22.46 mV Table 1. From its single-trimer conductance (0.53 nS at 50 mM NH₄Cl) and the fitted permeability ratio (P_Cl⁻∕P_NH₄⁺ ≈ 5∶1), the flux rate was calculated to roughly ~6000 NH₄⁺·s⁻¹·monomer⁻¹ at a 1 µM transmembrane NH₄⁺ gradient. This rate indicates that, even under low micromolar conditions, Omp-Pst2 demonstrated efficient NH₄⁺ entry. Substituting chloride with sulfate (ammonium sulfate: 200 mM cis∕50 mM trans) modestly reduced the driving force (V_rev = 20.06) and increased the anion-to-cation permeability ratio (P_SO₄²⁻∕P_NH₄⁺ ≈ 6∶1), yielding an extrapolated flux of ~5500 NH₄⁺·s⁻¹·monomer⁻¹ at 1 µM. The roughly ~15% decrease in NH₄⁺ throughput when sulfate is the counterion might possibly be due to sulfate’s larger size and higher hydration energy [80,81], but Omp-Pst2 still maintains a comparatively high transport rate.

Under identical NH₄Cl conditions (200 mM cis ∕ 50 mM trans), OmpF produced V_rev = 18.5, with a fitted permeability ratio (P_Cl⁻∕P_NH₄⁺ ≈ 9∶1). Using its single-trimer conductance (0.40 nS at 50 mM NH₄Cl), this translates into an extrapolated NH₄⁺ flux of ~4000 NH₄⁺·s⁻¹·monomer⁻¹ at 1 µM—roughly 30% lower than Omp-Pst2 under the same conditions. When ammonium sulfate was substituted (200 mM cis ∕ 50 mM trans), V_rev remained around 18 mV and the P_SO₄²⁻∕P_NH₄⁺ ratio stayed near 9∶1, giving a slightly higher flux (~5000 NH₄⁺·s⁻¹·monomer⁻¹ at 1 µM) but still in a same range of Omp-Pst2’s flux rates. Structurally, Omp-Pst2’s constriction zone is marginally wider and lined with acidic residues that create an electrostatic environment conducive to NH₄⁺ passage with moderate anion exclusion. By contrast, OmpF’s tighter eyelet and more densely arranged charges trade flux for discriminative power: its permeability ratio against anions is ~9∶1 anion to cation for NH₄⁺ but the maximum NH₄⁺ flux is only ~70–80% of what Omp-Pst2 achieves. These observations underscore how pore diameter, residue distribution, and local electrostatics balance throughput versus selectivity in general diffusion porins [82,83].

Biologically, passive NH₄⁺ diffusion through general porins complements active transport under nutrient-limited conditions [7,19,63,65]. Extracellular NH₄⁺ often falls to low micromolar levels in soils, aquatic microniches, or host niches such as the urinary tract [3,74,84]. In such contexts, energy-intensive transporters (e.g., AmtB) may be downregulated or saturated, whereas porin-mediated entry provides an energy-efficient entry route [5,6,7]. The extrapolated rates (~5000–6000 NH₄⁺·s⁻¹·monomer⁻¹ via Omp-Pst2 vs. ~4000–5000 via OmpF at 1 µM) suggest that Providencia stuartii can sustain periplasmic NH₄⁺ levels through Omp-Pst2 under low-nitrogen conditions, while Escherichia coli OmpF’s tighter selectivity may prioritize ionic homeostasis over maximal NH₄⁺ throughput. Switching the counterion from Cl⁻ to SO₄²⁻ fluctuates NH₄⁺ flux in both porins, highlighting how extracellular anion composition could modulate nutrient uptake efficiency. From an antimicrobial-strategy perspective, disrupting high-flux channels like Omp-Pst2 could hinder bacterial growth under nitrogen scarcity, whereas leveraging Omp-Pst2’s broad NH₄⁺ permeability might aid in designing NH₄⁺-mimicking prodrugs. In contrast, OmpF’s strict selectivity narrows the range of permeable molecules but ensures tighter control of periplasmic ionic balance. Overall, these quantitative measurements of NH₄⁺ transport reveal a delicate balance between efficiency and selectivity in bacterial outer membrane porins and highlight potential avenues for targeting nutrient uptake pathways in Gram-negative pathogens.

7. Conclusions

This study employed a simple and dependable electrophysiological technique [35,36] to quantify the transport of ammonium through two bacterial outer membrane porins: Omp-Pst2 from Providencia stuartii and OmpF from Escherichia coli [24,28,36]. Forming an artificial lipid bilayer and applying controlled salt gradients [36] allowed us to record the electrical currents generated by individual porin channels, revealing several key insights [36].

(1)

Higher ammonium flux through Omp-Pst2

Under identical ammonium chloride gradients, Omp-Pst2 conducted approximately ≈ 5000-6000 ammonium ions per second, whereas flux from OmpF conducted around ≈ 4000-5000 ions per second. Even when ammonium sulfate was used, Omp-Pst2 maintained a higher overall flux than OmpF. These results suggest that in environments with limited ammonium [1,62,85], Omp-Pst2 can deliver nitrogen to the periplasm faster than OmpF.

(2)

Greater selectivity in OmpF

Although OmpF transported fewer ammonium ions overall, it did so with a stronger preference for ammonium over its counterion (chloride or sulfate). In contrast, Omp-Pst2 allowed a higher proportion of counterions to pass alongside ammonium. This suggests that OmpF may play a more pronounced role in maintaining ionic balance within the periplasm [86] while Omp-Pst2 focuses on maximizing throughput.

(3)

Influence of counterions

Switching the accompanying anion from chloride to sulfate altered the transport speed and selectivity of each porin. Specifically, sulfate reduced ammonium flux slightly compared to chloride. This observation highlights the fact that the mixture of available salts in real bacterial cells can directly influence the efficiency with which porins import ammonium. Taken together, these results shed light on how Gram-negative bacteria adjust their outer-membrane channels to optimize nitrogen uptake under different environmental conditions [1,58,62]. Omp-Pst2 is especially well-suited for rapid ammonium delivery, which could be advantageous for Providencia stuartii growing in nutrient-poor environments, such as a catheterized urinary tract [86,87]. Conversely, OmpF’s stronger selectivity may help Escherichia coli maintain periplasmic ionic balance in more variable habitats [15,88,89,90,91,92]. Understanding these complementary roles is important for both basic microbiology and identifying potential weak points in bacterial nutrient acquisition [15,16,22,32,88,89,90,91,92]. For instance, blocking or misdirecting a porin that typically supplies ammonium may hinder a pathogen’s ability to thrive in nitrogen-limited environments [85,93,94].

(4)

Limitations

While these findings are encouraging, several factors must be considered when interpreting the results:

(1)

Simplified Bilayer Model

All experiments were conducted in a purified, laboratory-made lipid bilayer. Real bacterial outer membranes are much more complex—they contain lipopolysaccharides, various lipids, and other proteins that can influence how porins behave. As a result, the transport rates measured here may differ from those in an actual bacterial cell [13,15,91,92,95,96,97,98].

(2)

Absence of Cellular Context

In living bacteria, porin levels are regulated in response to environmental signals, and other proteins (such as periplasmic binding proteins) may work together with porins to optimize ammonium uptake. None of these regulatory or cooperative factors were present in the bilayer system, so the “pure” porin behavior described here may not fully capture what happens in vivo [17,21,61,99,100,101].

(3)

Limited Porin Selection

This study focuses on just two general diffusion porins, Omp-Pst2 and OmpF. Many other porins exist in different bacterial species, each with its own transport characteristics. Therefore, one should be cautious about generalizing these findings to all Gram-negative bacteria [13,15,22,48,61,91,92,96,97,98,102,103].

(4)

Fixed Experimental Conditions

The salt concentrations, pH, and temperature used here were chosen for consistency and reproducibility. However, bacterial cells often experience a wider range of pH values and ionic strengths. It remains to be seen how Omp-Pst2 and OmpF would perform under those varying conditions [15,91,95,96,104,105].

(5)

Lack of Structural and Computational Insights

Although I discuss how differences in pore size, charge distribution, and lining residues may explain why Omp-Pst2 carries more ammonium while OmpF is more selective, no detailed structural modeling or molecular dynamics simulations were performed. As a result, the precise atomic-level reasons for the observed behaviors remain speculative [17,21,54,106,107,108].

(6)

Future Directions

To build on these results, future work could include the following:

(1)

In Vivo Validation

Measuring ammonium uptake rates in living bacterial cultures that express only one porin at a time would help confirm whether the trends seen in the bilayer hold true inside cells [13,15,68,90,91,92,109,110,111].

(2)

Broader Porin Survey

Testing additional porins—especially those found in other clinically relevant pathogens—would clarify whether the high throughput of Omp-Pst2 is unique or part of a wider pattern [88,111,112].

(3)

Structural and Computational Studies

High-resolution structures (e.g., from X-ray crystallography or cryo-EM) and computer simulations could reveal exactly how specific amino acids and pore geometry determine both flux and selectivity [50,71,107,108,113,114].

(4)

Antimicrobial Testing

Small molecules or peptides designed to block Omp-Pst2 could be screened for their ability to inhibit growth under nitrogen-limited conditions. Similarly, exploring drug conjugates that exploit these porins as “entry points” might open new paths for delivering antibiotics more effectively [48,102,103].

By addressing these limitations and pursuing these follow-up studies, a more complete picture will emerge of how outer membrane porins contribute to bacterial survival, nutrient acquisition, and potential vulnerabilities that can be targeted in antimicrobial therapy.