## 5 Materials and Methods
### 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 \(P_{cation}/P_{anion}\):  
\[\Delta V=RT/F\times ln\left(\frac{P_{cation}\times[cation]^{cis}+P_{anion} \times[anion]^{trans}}{P_{cation}\times[cation]^{trans}+P_{anion}\times[ anion]^{cis}}\right) \tag{1}\]  
where \(\Delta V\) is the measured reversal potential, \(P_{\text{cat}}\) 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 10\({}^{4}\) 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 1\(\upmu\)M 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 \(\approx\) (1:5), correlating with a higher ion flux rate. In contrast, OmpF shows a much higher permeability ratio \(\approx\) (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.