Role of Electroosmosis in the Permeation of Neutral Molecules: CymA and Cyclodextrin as an Example

Abstract

To quantify the flow of small uncharged molecules into and across nanopores, one often uses ion currents. The respective ion-current fluctuations caused by the presence of the analyte make it possible to draw some conclusions about the direction and magnitude of the analyte flow. However, often this flow appears to be asymmetric with respect to the applied voltage. As a possible reason for this asymmetry, we identified the electroosmotic flow (EOF), which is the water transport associated with ions driven by the external transmembrane voltage. As an example, we quantify the contribution of the EOF through a nanopore by investigating the permeation of α-cyclodextrin through CymA, a cyclodextrin-specific channel from Klebsiella oxytoca. To understand the results from electrophysiology on a molecular level, all-atom molecular dynamics simulations are used to detail the effect of the EOF on substrate entry to and exit from a CymA channel in which the N-terminus has been deleted. The combined experimental and computational results strongly suggest that one needs to account for the significant contribution of the EOF when analyzing the penetration of cyclodextrins through the CymA pore. This example study at the same time points to the more general finding that the EOF needs to be considered in translocation studies of neutral molecules and, at least in many cases, should be able to help in discriminating between translocation and binding events.

Identification and Functional Characterization of a Novel OprD-like Chitin Uptake Channel in Non-chitinolytic Bacteria

“…Our BLM data showed that EcChiP interacted strongly with long-chain chitooligosaccharides but not with maltooligosaccharides, implying that the channel was specific for chitooligosaccharide uptake. Strong interaction with the higher molecular weight substrates is also a characteristic of other sugar-specific channels, such as LamB (37,38,49), VhChiP (8,48), and CymA (50). Consistent with this is an earlier in vivo study that showed no growth of S. marcescens expressingthenullChiPmutantinthepresenceofchitooligosaccharides larger than chitotriose (26).…”

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

“…Thes ingle-channel measurement was carried out in the presence of 0.2 mm norfloxacin. In as imilar analysis we obtained the voltage-dependent association rate and dwell time (Figure 3) [18,20] Increasing negative voltages increases k on in OmpF wt from 1.3 10 4 to 1.8 10 5 m À1 s À1 in an exponential [a] flux at 1mm Arginine-Cl gradient was determined.…”

Permeation von kleinen Molekülen durch Membrankanäle: Chemische Modifikation zur Quantifizierung des Transports über OmpF

“…Mit einer ähnlichen Analyse wie oben erhielten wir die spannungsabhängige Assoziationsrate und die Aufenthaltszeiten (Abbildung 3). [18,20] In OmpF wt führen steigende negative Spannungen zu einem exponentiellen Anstieg der Assoziationsrate von 1.3 10 4 auf 1.8 10 5 m À1 s À1 ,w ährend die Verweildauer 700 msa uf 60 msl inear abnimmt. Die OmpF E181C MTSES Mutante ergab einen ähnlichen Tr end bezüglich der Spannungsabhängigkeit der Assoziationsrate (5 10 3 bis 2.5 10 6 m À1 s À1 [a] Fluss umgerechnet auf 1mm Arginin-Cl-Gradient.…”

“…[18,20] In OmpF wt führen steigende negative Spannungen zu einem exponentiellen Anstieg der Assoziationsrate von 1.3 10 4 auf 1.8 10 5 m À1 s À1 ,w ährend die Verweildauer 700 msa uf 60 msl inear abnimmt. Mit einer ähnlichen Analyse wie oben erhielten wir die spannungsabhängige Assoziationsrate und die Aufenthaltszeiten (Abbildung 3).…”