Biophys J, October 1999, p. 2003-2014, Vol. 77, No. 4

Membrane Fusion and the Lamellar-to-Inverted-Hexagonal Phase Transition in Cardiolipin Vesicle Systems Induced by Divalent Cations

 ^*Department of Physiological Chemistry, University of Groningen, 9713 AV Groningen, and Departments of  ^#Biochemistry of Membranes and  ^§Molecular Cell Biology, University of Utrecht, 3584 CH Utrecht, The Netherlands

The polymorphic phase behavior of bovine heart cardiolipin (CL) in the presence of different divalent cations and the kinetics of CL vesicle fusion induced by these cations have been investigated. ³¹P-NMR measurements of equilibrium cation-CL complexes showed the lamellar-to-hexagonal (L-H_II) transition temperature (T_H) to be 20-25°C for the Sr²⁺ and Ba²⁺ complexes, whereas in the presence of Ca²⁺ or Mg²⁺ the T_H was below 0°C. In the presence of Sr²⁺ or Ba²⁺, CL large unilamellar vesicles (LUVs) (0.1 µm diameter) showed kinetics of destabilization, as assessed by determination of the release of an aqueous fluorescent dye, which strongly correlated with the L-H_II transition of the final complex: at temperatures above the T_H, fast and extensive leakage, mediated by vesicle-vesicle contact, was observed. On the other hand, mixing of vesicle contents was limited and of a highly transient nature. A different behavior was observed with Ca²⁺ or Mg²⁺: in the temperature range of 0-50°C, where the H_II configuration is the thermodynamically favored phase, relatively nonleaky fusion of the vesicles occurred. Furthermore, with increasing temperature the rate and extent of leakage decreased, with a concomitant increase in fusion. Fluorescence measurements, involving incorporation of N-NBD-phosphatidylethanolamine in the vesicle bilayer, demonstrated a relative delay in the L-H_II phase transition of the CL vesicle system in the presence of Ca²⁺. Freeze-fracture electron microscopy of CL LUV interaction products revealed the exclusive formation of H_II tubes in the case of Sr²⁺, whereas with Ca²⁺ large fused vesicles next to H_II tubes were seen. The extent of binding of Ca²⁺ to CL in the lamellar phase, saturating at a binding ratio of 0.35 Ca²⁺ per CL, was close to that observed for Sr²⁺ and Ba²⁺. It is concluded that CL LUVs in the presence of Ca²⁺ undergo a transition that favors nonleaky fusion of the vesicles over rapid collapse into H_II structures, despite the fact that the equilibrium Ca²⁺-CL complex is in the H_II phase. On the other hand, in the presence of Sr²⁺ or Ba²⁺ at temperatures above the T_H of the respective cation-CL complexes, CL LUVs rapidly convert to H_II structures with a concomitant loss of vesicular integrity. This suggests that the nature of the final cation-lipid complex does not primarily determine whether CL vesicles exposed to the cation will initially undergo a nonleaky fusion event or collapse into nonvesicular structures.

Biophys J, October 1999, p. 2003-2014, Vol. 77, No. 4
© 1999 by the Biophysical Society   0006-3495/99/10/2003/12  $2.00

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