Cardiolipin-cholesterol interactions in the liquid-crystalline phase: a steady-state and time-resolved fluorescence anisotropy study with cis- and trans-parinaric acids as probes.

The potency of cholesterol to affect the acyl chain order and dynamics of cardiolipin membranes in the liquid-crystalline state was monitored by steady-state and time-resolved fluorescence anisotropy as well as excited-state lifetime measurements with cis- and trans-parinaric acids as probes. Up to a cholesterol mole fraction (mean chl) of congruent to 0.20, no measurable effect on any of the fluorescence parameters of either probe in cardiolipin bilayers was evidenced. This was in striking contrast to the situation in dioleoylphosphatidylcholine (DOPC), for which a cholesterol mole fraction of 0.20 corresponded to the half-maximal effect on the fluorescence parameters, reflecting the classical ordering effect of cholesterol observed in lecithin systems in the liquid-crystalline phase. Whereas in DOPC bilayers this order effect plateaued at mean chl = 0.50, in cardiolipins the increase in acyl chain order was observable up to a mole fraction as high as 0.80. This indicated that cardiolipins were able to incorporate about 4 mol of cholesterol/mol of cardiolipin (i.e., 1 mol of cholesterol per fatty acyl chain). Besides, 31P NMR spectra of multilamellar liposomes obtained from pure cardiolipins and cardiolipin--cholesterol mixtures evidenced a line shape characteristic of lamellar structures. These results clearly indicate that the presence of high levels of cardiolipins in inner mitochondrial membrane does not impede cholesterol uptake by these membranes. However, the absence of an effect of cholesterol in the physiological range of the cholesterol mole fraction (congruent to 0.20) would signify weaker sterol-cardiolipin interactions than with lecithins and in turn would explain the relative dearth of cholesterol in these membranes.