Membrane fusion and the lamellar-to-inverted-hexagonal phase transition incardiolipin vesicle systems induced by divalent cations

The polymorphic phase behavior of bovine heart cardiolipin (CL) in the pres ence of different divalent cations and the kinetics of CL vesicle fusion in duced by these cations have been investigated. P-31-NMR measurements of equ ilibrium cation-CL complexes showed the lamellar-to-hexagonal (L-alpha-H-11 ) transition temperature (T-H) to be 20-25 degrees C for the Sr2+ and Ba2complexes, whereas in the presence of Ca2+ or Mg2+ the T-H was below 0 degr ees C. In the presence of Sr2+ or Ba2+, CL large unilamellar vesicles (LUVs ) (0.1 mu m diameter) showed kinetics of destabilization, as assessed by de termination of the release of an aqueous fluorescent dye, which strongly co rrelated with the L-alpha-H-11 transition of the final complex: at temperat ures above the T-H fast and extensive leakage, mediated by vesicle-vesicle contact, was observed, On the other hand, mixing of vesicle contents was li mited and of a highly transient nature. A different behavior was observed w ith Ca2+ or Mg2+: in the temperature range of 0-50 degrees C, where the H-1 1 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 fu sion. Fluorescence measurements, involving incorporation of N-NBD-phosphati dylethanolamine in the vesicle bilayer, demonstrated a relative delay in th e L-alpha-H-11 phase transition of the CL vesicle system in the presence of Ca2+. Freeze-fracture electron microscopy of CL LUV interaction products r evealed the exclusive formation of H-11 tubes in the case of Sr2+, whereas with Ca2+ large fused vesicles next to H-11 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 co ncluded that CL LUVs in the presence of Ca2+ undergo a transition that favo rs nonleaky fusion of the vesicles over rapid collapse into H-11 structures , despite the fact that the equilibrium Ca2+-CL complex is in the H-11 phas e. 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-11 structures with a concomitant loss of vesicular integrity. This sugges ts that the nature of the final cation-lipid complex does not primarily det ermine whether CL vesicles exposed to the cation will initially undergo a n onleaky fusion event or collapse into nonvesicular structures.