Mitochondrial Protein Import Channels

General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpF

“…9−11 We followed a previous suggestion to analyze the selectivity of the OmpF containing artificial bilayer membranes under bi-and triionic conditions on both sites of the planar bilayer. 5,11,12 Because ion fluxes are also created by concentration gradients, the GHK current equation allows calculation of the relative ion permeability by macroscopic GHK theory using the chemical potential created by the different electrophoretic mobility of the ions themselves. 9 Starting from the GHK current equation for a given composition of ions in solution, the voltage-dependent total ion current I(V) crossing a membrane channel is given by the sum of the individual ion currents…”

“…The measurement of the ion selectivity of a membrane pore is an established method to obtain the relative permeability in terms of fluxes of the ions present in solution. However, in order to determine turnover numbers of the individual permeating ions, knowledge of their single-channel conductances is also required. − Here we describe an approach to quantify the permeation of three charged β-lactamase inhibitors, namely, avibactam, sulbactam, and tazobactam (Figure S1), through the OmpF porin from Escherichia coli. Moreover, we show that the macroscopic turnover number obtained experimentally can be complemented with all-atom molecular dynamics (MD) modeling, providing atomic details on the selectivity and the energetics of transport.…”

“…Single-Channel Recording under Asymmetric Conditions: Bi- and Tri-ionic Potential. In order to gain information on the permeability of OmpF for the β-lactamase inhibitor anions, we applied an alternative experimental approach based on the Goldman–Hodgkin–Katz (GHK) current equation. − We followed a previous suggestion to analyze the selectivity of the OmpF containing artificial bilayer membranes under bi- and tri-ionic conditions on both sites of the planar bilayer. ,, Because ion fluxes are also created by concentration gradients, the GHK current equation allows calculation of the relative ion permeability by macroscopic GHK theory using the chemical potential created by the different electrophoretic mobility of the ions themselves . Starting from the GHK current equation for a given composition of ions in solution, the voltage-dependent total ion current I ( V ) crossing a membrane channel is given by the sum of the individual ion currents I x ( V ) and specifically in our case the main contributors are Na + , Cl – , and the inhibitor (inh – )­ with where V is the transmembrane voltage, P x the permeability for the ion x, z the valency, F = 9.6 × 10 4 As mol –1 the Faraday constant, and R = 8.3 J mol –1 K –1 the gas constant.…”

“…In particular, with nanometer sized pores at low mM concentrations of current carrier electrolytes like NaCl, the nanopore permeability of large charged solute molecules like the β-lactamase inhibitor anions of avibactam, sulbactam, and tazobactam can be screened under tri-ionic conditions using low mM to μM concentrations of the charged large solute molecules. Besides the principal difficulties underlying the GHK constant field theory, which assumes independent movement of the ions through the pores (see refs − , for a detailed discussion), we have demonstrated that the methodology can be used to obtain semiquantitative measures for permeation of charged drugs through nanopores. The method presented can, after a suitable miniaturization and parallelization, serve as a basis for a simple, fast, and sensitive permeability screen of nanopores for charged molecules.…”