title: 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.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Kuyper
authors.forename: Jason S
authors.initials: JS
authors.lastname: Kuo
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Mutch
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Chiu
mesh_terms: D002463:Cell Membrane Permeability; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000379:methods; D008856:Microscopy, Fluorescence; D010713:Phosphatidylcholines / Q000737:chemistry*; D011522:Protons
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D011522:Protons; C024098:6-carboxyfluorescein; C028694:1-palmitoyl-2-oleoylphosphatidylcholine