The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Abstract

Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2'-chloro,4'-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as "carriers" for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.

The mechanism of action of DNP on phospholipid bilayer membranes

“…where o-is the charge density in electronic charges per A 2, C is the total monovalent ion concentration in the bulk solution in moles per liter and RT]F= 25.3 mV at 22 ~ The data of Hopfer et al (1970) illustrate, however, that the ratio of the conductances produced by DNP on a neutral and positive bilayer membrane is much less than that predicte& by the combination of Eqs. (8) and (9) and, furthermore, that the ratio depends markedly on the pH.…”

“…(7) that irrespective of the value of ~ the conductance should attain a maximum value when [H § =K1. Hopfer et al (1970) observed, however, that the conductance maximum was shifted about a pH unit to the acid side of the pK when measurements were made with 10-4M DNP on a bilayer formed from a positive lipid. Next, Eq.…”

“…Lea and Croghan (1969) examined experimentally the properties of a bilayer exposed to 1 mM DNP and concluded that the permeant species with this uncoupler was also the HA I complex. More recently, however, Hopfer et al (1970) have obtained data with DNP on positive, neutral and negative membranes which appear to contradict the carrier model. They noted that if the carrier model is correct, maxima sould occur in the conductance vs. pH curves at the pK of the weak acid irrespective of the charge on the membrane and that the difference in the conductance observed on a neutral membrane and one with a constant surface charge should be independent of pH.…”

“…Although its mechanism of action on these biological systems is unknown, some innovative support for the chemiosmotic hypothesis of Mitchell (1966) was obtained by Bielawski, Thompson and Lehninger (1966), who demonstrated that DNP enhances the conductance of a phospholipid bilayer. Many other uncouplers have since been shown to also increase the conductance of phospholipid membranes (e.g., Skulachev, Sharaf & Liberman, 1967;Hopfer, Lehninger & Thompson, 1968;Liberman & Babakov, 1968;Liberman, Mochova, Skulachev & Topaly, 1968;Liberman & Topaly, 1968 a, b;Hopfer, Lehninger & Lennarz, 1970) but all attempts to formulate a model which would explain the effects of the uncouplers on bilayers in terms of a single permeation mechanism have met with a singularlack of success. The main reason for this, as LeBlanc (1971) has recently pointed out, is that there must be at least two classes of weak acid uncouplers and two different mechanisms of charge permeation.…”

Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1

“…The mechanism of action of these agents is most simply explained in terms of their well-documented ability to increase proton conductance across model membrane systems (5,6) and t o inhibit energy-linked proton movement in energytransducing membranes (e.g., Ref. 7).…”