Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous

Abstract

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.

Roles of Carboxyl Groups in the Transmembrane Insertion of Peptides

“…When we varied the pH of the solution outside the liposomes, the fluorescence of the encapsulated probe changed in a sigmoid fashion, with an apparent pKa of 5.05 (data not shown). A relatively high proton permeation through unilamellar POPC liposomes in the minute time-scale has been reported elsewhere 22, 23 . On the other hand, our kinetics data suggest that the time of wt peptide exit (with 2 TM and 4 C-terminal protonatable groups) is in the range of milliseconds 6 .…”

pH dependent transfer of nano-pores into membrane of cancer cells to induce apoptosis

“…It led to the quenching of fluorescence of FITC located at the inner leaflet of bilayer (red line on Figure 2c). The data are in an accordance with the previously published results1920. At the same time, when 2% or 10% of gA was incorporated into liposomes the decrease of FITC fluorescence was completed within less than 5 ms due to the conductance of protons by the gA channel (blue line on Figure 2c).…”

Determining the number of specific proteins in cellular compartments by quantitative microscopy

“…The use of TIRF greatly reduces background fluorescence by illuminating only ∼300 nm above the coverslip, thereby increasing the signal-to-noise ratio of the image 13 . The details of setting up a two-color TIRF microscope are beyond the scope of this protocol, and can be found elsewhere 13,14 . The two lasers chosen for the experiment must be wavelength matched with the fluorophores used, and they must be stable over the time-course of the experiment.…”