Quantitative visualization of passive transport across bilayer lipid membranes

Abstract

The ability to predict and interpret membrane permeation coefficients is of critical importance, particularly because passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. We present a method for the quantitative measurement of the permeation coefficients of protonophores by using laser confocal scanning microscopy coupled to microelectrochemistry, which is amenable to precise modeling with the finite element method. The technique delivers well defined and high mass transport rates and allows rapid visualization of the entire pH distribution on both the cis and trans side of model bilayer lipid membranes (BLMs). A homologous series of carboxylic acids was investigated as probe molecules for BLMs composed of soybean phosphatidylcholine. Significantly, the permeation coefficient decreased with acyl tail length contrary to previous work and to Overton's rule. The reasons for this difference are considered, and we suggest that the applicability of Overton's rule requires re-evaluation.Overton's rule ͉ permeation ͉ ultramicroelectrode ͉ laser confocal scanning microscopy ͉ finite element method

A new approach for the fabrication of microscale lipid bilayers at glass pipets: application to quantitative passive permeation visualization

“…Moreover, for membranes formed 55 from soy or egg PC lipids, the paintbrush method has commonly been used to form bilayers, leaving residual organic solvent molecules within the bilayers which may significantly impact on the rate of transport. This may explain the rather weak relationship between P and K seen in our earlier study 1 The DPPC data obtained in the present study also show a strong linear trend between P and K with the exception of acetic acid, where the P value is lower than would be expected based on Overton's rule. This observation could be attributed to the gel 65 phase structure of the bilayer creating a greater energy barrier for the permeation of the more hydrophilic molecule, compared to those with longer acyl tails.…”

“…Using this system, we determine the permeation coefficients of a series of aliphatic carboxylic acids as 35 they passively permeate across a bilayer. By using CLSM with a pH-sensitive fluorophore, 1,16 the movement of these molecules can be tracked by monitoring the local pH changes around the end of the pipet. Combining this with FEM modeling, permeation coefficients for the series of acids can be extracted to determine 40 the effect of permeant lipophilicity on permeability, using just one adjustable parameter (permeation coefficient) to model the data Significantly, the trends observed are in quantitative agreement with measurements on solvent-free liposomes, confirming the validity of the technique.…”

“…The protonation state of fluorescein has been shown to have no significant effect at the concentrations 50 used here and was therefore ignored in the calculations. 1 The very fast kinetics of the protonation processes compared to the experimental timescale mean that they could be considered as equilibria controlled by the local pH. To ensure the equilibria were handled correctly, the pKa values for the weak acid and buffer were corrected for ionic activity using the Davies equation.…”

Windows to cell function and dysfunction: Signatures written in the boundary layers

“…(9,10) Even under extreme conditions of mixing, levels which would destroy most cells, this layer remains at approximately 0.7m. (11) Within the USL diffusion dominates solute transfer and is defined by the Stokes-Einstein (Sutherland-Einstein) equation such that:…”

“…Whereas Overton's Rule would dictate that the partition coefficient into the membrane is the primary determinant of the permeation coefficient recent work brings this long standing rule into doubt. (11) The USL itself will impact significantly depending upon the solute (11) . Where the membrane forms an impermeable layer, the activity in the USL approximates that of the bulk solution.…”