Biological channels facilitate the exchange of
small molecules across membranes, but surprisingly there is a lack of general
tools for the identification and quantification of transport (i.e.,
translocation and binding). Analyzing the ion current fluctuation of a typical
channel with its constriction region in the middle does not allow a direct
conclusion on successful transport. For this, we created an
additional barrier acting as a molecular counter at the exit of the channel. To
identify permeation, we mainly read the molecule residence time in the channel
lumen as the indicator whether the molecule reached the exit of the channel. As
an example, here we use the well-studied porin, OmpF, an outer membrane channel
from <i>E. coli</i>. Inspection of the
channel structure suggests that aspartic acid at position 181 is located below
the constriction region (CR) and we subsequently mutated this residue to
cysteine, where else cysteine free and functionalized it by covalent binding
with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione
(GLT) blockers. Using the dwell time as the signal for transport, we found that
both mono-arginine and tri-arginine permeation process is prolonged by 20% and
50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized
blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the
permeation of mono-arginine by 9-fold and even blocked the pathway of the
tri-arginine. In case of the hepta-arginine as substrate, both chemical
modifications led to an identical ‘blocked’ pattern observed by the dwell time
of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for
antibiotic permeation, we analyzed norfloxacin, a
fluoroquinolone antimicrobial agent. The
modulation of the interaction dwell time suggests possible successful
permeation of norfloxacin across OmpF<sub>wt</sub>.
This approach may discriminate blockages from translocation events for a wide
range of substrates. A potential application could be screening for scaffolds
to improve the permeability of antibiotics.
Capturing single molecules by nanopores: measured times and thermodynamics