Kinetics for development of gramicidin-induced ion permeability in unilamellar phospholipid vesicles.

Two kinetics for the development of gramicidin-dependent cation permeability in small unilamellar vesicles have been studied by using a vesicle-entrapped, pH-sensitive fluorescence probe to continuously report changes in intravesicular pH. The incorporation of 4-5 gramicidin dimers/vesicle was sufficient to increase the proton and counterion permeability of that vesicle by several orders of magnitude, so that ionic equilibration following a perturbation of the external medium pH occurred in less than 1 s. Once a functional gramicidin dimer has become incorporated into one vesicle, it does not readily exchange into another, so that the effects of gramicidin with regard to an individual vesicle can be considered to be essentially "all or none." The rate at which transmembrane ion permeability develops in a vesicle suspension was found to depend upon the degree of fluidity of the membrane hydrocarbon interior, being much lower at low temperatures or when cholesterol was present in the bilayer. Low temperatures and increasing bilayer cholesterol content also decreased the number of vesicles affected by a given gramicidin concentration, indicating a decreased membrane solubility for the ionophore at low bilayer fluidities.

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles.

The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.

Modulation by small hydrophobic molecules of valinomycin-mediated potassium transport across phospholipid vesicle membranes.

The effects of small hydrophobic molecules on valinomycin-mediated K+ transport in small unilamellar soybean phospholipid vesicles have been studied by using a vesicle-entrapped pH-sensitive hydrophilic fluorescence probe to monitor counterion-limited, passive H+ diffusion into vesicles after an abrupt decrease in external pH [Clement, N. R., & Gould, J. M. (1981) Biochemistry (preceding paper in this issue)]. Under conditions where, even in the absence of valinomycin, transmembrane KL+ movement represented the primary and limiting counterion flux, less than 1 valinomycin molecule/vesicle was sufficient to accelerate the rate of H+ entry into all of the vesicles. Incorporation of the bulkily substituted molecules butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and p-di-tert-butylbenzene into soybean lipid bilayers had no effect upon K+ diffusion in the absence of valinomycin. However, the presence of these hydrophobic molecules increased the apparent efficacy for K+ transport of a given valinomycin concentration by as much as 4-6 fold. The less bulky membrane perturbants tert-butyl alcohol, phenol, and heptane showed very much less dramatic effects. While the rate of valinomycin-mediated K+ transport (in the presence or absence of BHT) was very sensitive to temperature-induced changes in membrane fluidity, the degree of synergistic interaction between valinomycin and BHT was independent of temperature. Furthermore, BHT, BHA, and p-di-tert-butylbenzene, at levels which alter valinomycin-mediated K+ transport, did not by themselves induce changes in membrane fluidity. It is postulated that changes in phospholipid head-group packing and/or surface charge density brought about by the presence of bulky perturber molecules leads to changes in partitioning of valinomycin or the valinomycin-K+ complex between the aqueous and membrane phases.