Small‐Molecule Permeation across Membrane Channels: Chemical Modification to Quantify Transport across OmpF

A molecule counter, added at the channel exit of a bacterial cell membrane pore creates an additional energy barrier at the to quantify molecules that permeate. The efficiency with charged peptides was tested. In a second series the antibiotic norfloxacin was tested. Introducing the partial channel blocker allows to distinguish binding from translocation for a broad range of molecules.

Abstract

Biological channels facilitate the exchange of molecules across membranes, but general tools to quantify transport are missing. Electrophysiology is the method of choice to study the functional properties of channels. However, analyzing the current fluctuation of channels typically does not identify successful transport, that is, distinguishing translocation from binding. To distinguish both processes, we added an additional barrier at the channel exit acting as a molecular counter. To identify permeation, we compare the molecule residence time in the native channel with one that is chemically modified at the exit. We use the well‐studied outer membrane channel from E. coli, OmpF. Position 181, which is below the constriction region, was subsequently mutated into cysteine (E181C) in an otherwise cysteine‐free system, then functionalized by covalent binding with one of the two blockers MTSES or GLT. We measured the passage of model peptides, mono‐, tri‐, hepta‐arginine and of norfloxacin, as an example for antibiotic permeation.

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