Aluminum-induced lipid phase separation and membrane fusion does not require the presence of negatively charged phospholipids.

The interaction of Aluminum with phosphatidyl serine lipid vesicles containing variable amounts of phosphatidyl ethanolamine, phosphatidyl choline and cholesterol has been studied by lipid phase separation monitored by fluorescence quenching. The interaction of Al3+ with neutral phospholipid membranes has also been investigated. Maximal lipid phase separation can be demonstrated in mixed phosphatidyl ethanolamine-cholesterol vesicles when using concentrations of aluminum between 87.5 and 125 microM. Millimolar concentrations of Ca2+, Mn2+, Cd2+ and Zn2+ were without any effect. Aluminum also induced fusion of phospholipid membranes monitored by resonance energy transfer between N-(7-nitro-2,1,3, benzoxadiazol-4 yl) phosphatidyl ethanolamine and N-(lissamine Rhodamine B-sulfonyl) phosphatidyl ethanolamine, either when containing low amounts of phosphatidyl serine (12.5%) or without any negatively charged phospholipid. Aluminum-induced fusion of liposomes was also monitored by the fluorescence of the terbium-dipicolinic acid complex (Tb-DPA3-) formed during fusion of vesicles containing either Tb-(citrate)6- complex or sodium salt of dipicolinic acid.

Synergistic effects of micromolar concentrations of Zn2+ and Ca2+ on membrane fusion.

Resonance Energy Transfer between N-(7-nitro-2,1,3 benzoxadiazol -4 yl) phosphatidyl ethanolamine and N-Lissamine-Rhodamine B sulfonyl) phosphatidyl ethanolamine embedded in two different populations of small unilamellar vesicles made of phosphatidyl serine has been used to study the fusion process induced by Zn2+ and Ca2+. Lipid intermixing demonstrating fusion of liposome membranes can already be observed at 125 and 250 mumol/l of Zn2+. After short time pre-incubations with micromolar concentrations of Zn2+ as low as 150 mumol/l, Ca2+ induces an instantaneous increase of vesicle fusion. The lipid intermixing induced by micromolar concentrations of Ca2+ (250-500 mumol/l) could be increased up to 4 times when pre-incubated with 150 or 200 mumol/l of Zn2+. The effect of 1 mM of Ca2+ alone on lipid intermixing can be mimicked by 150 mumol/l of Zn2+ followed by 500 mumol/l of Ca2+. Our data demonstrate that Zn2+ and Ca2+ act synergistically to affect cation-induced membrane fusion. We suggest that Zn2+ specifically alters the physical state of phospholipid membranes making them more prone to calcium-triggered fusion.

Micromolar concentrations of Zn2+ potentiates Ca2+-induced phase separation of phosphatidyl serine containing liposomes.

Fluorescence quenching of 1-acyl-2-[6[(7 nitro-2,1,3-benzoxadiazol-4yl) amino]caproyl] phosphatidyl choline in small unilamellar vesicles consisting of phosphatidyl serine has been used to monitor the lipid phase separation induced by Zn2+ and Ca2+. Phase separation of vesicle membranes was observed with Zn2+ at concentrations as low as 125 microM. Low concentrations of Zn2+ required long incubation times to reach maximal quenching (120 minutes at 375 microM). When low concentrations of Ca2+ were added to the preparation during the developing phase of Zn2+-induced quenching, an explosive increase in fluorescence quenching was instantenously observed. Phase separation induced by sub-millimolar concentrations of Ca2+ could be increased at least 4 times when vesicles were pre-incubated with 250 microM of Zn2+.

Neurotoxic cations induce membrane rigidification and membrane fusion at micromolar concentrations.

The effect of the neurotoxic cations aluminum, cadmium and manganese on membranes was examined in sonicated unilamellar vesicles containing phosphatidylserine and compared to the effect of Ca2+. Fusion of membranes was monitored by assessing the resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-y)phosphatidylethanolamine and N-(lissamine-rhodamine B-sulfonyl)phosphatidylethanolamine. Self-quenching of high concentrations of carboxyfluorescein in liposomes was used to demonstrate the release of molecules entrapped in liposomes to compare the kinetics of leakage and intermixing of lipid. Rigidification of membranes was evaluated by monitoring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene embedded in membranes containing phosphatidylserine and dipalmitoylphosphatidylcholine. Cation-induced lipid intermixing of vesicles membranes and release of dye from the vesicles occurred in the same concentration range. With aluminum, these effects were observed with concentrations less than 25 microM. Significant rigidification of vesicle membranes was apparent with less than 25 microM of Al3+. Similar effects could only be observed with concentrations of Cd2+ and Mn2+ at least one order of magnitude higher (200 and 400 microM, respectively).

Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles.

The interaction of Al3+, Cd2+ and Mn2+ with phosphatidylserine-containing lipid vesicles was studied. Phase separation of vesicles was investigated by monitoring fluorescence quenching of the phospholipid analogue 1-palmitoyl-2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminocaproyl)phosphatidylcholine (C6-NBD-PC). Aggregation was determined by turbidimetry and leakage of vesicles content during fusion was monitored by the fluorescence of released 6-carboxyfluorescein. Al3+ demonstrated quenching at less than 30 mumol/l with a maximum effect at 100 mumol/l. Al3+-induced aggregation and dye release from the lipid vesicles were observed in the same concentration range. The effect of Cd2+ and Mn2+ on quenching was much less pronounced and could only be demonstrated in the 0.1-1 mmol/l range. Increasing amounts of phosphatidylcholine or phosphatidylethanolamine in the vesicles decreased both Al3+-induced quenching and aggregation, whereas cholesterol only slightly increased aggregation without affecting quenching.

Effect of lipid composition changes on carbocyanine dye fluorescent response.

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.