Deuteron NMR evidence for antibiotic-induced cholesterol immobilization in biological model membranes

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Abstract
The interaction of the polyene antibiotic filipin with membrane sterols was studied by deuterium NMR of the molecular probes [2,2,3,4,4,6-2H6]cholesterol and 1-myristoyl-2-[4'',4'',14'',14'',14''-2H5]myristoyl-sn-glycero-3-phosphocholine. At physiological temperatures, there is evidence of filipin-induced cholesterol immobilization in the membrane. The 2H NMR spectra of cholesterol show 2 domains in which ordering and dynamics are very different. In 1 of these, cholesterol is static on the 2H NMR time scale, whereas in the other it undergoes rapid axially symmetric motions similar to those it exhibits in the drug-free membrane: this indicates that the jumping frequency of cholesterol between the labile and immobilized domains is < 105/s. The distribution of cholesterol between these 2 sites is temperature dependent; at 0.degree. C all sterol molecules are immobilized, whereas at 60.degree. C they are almost totally in the labile site. The phospholipids sense only 1 type of environment, at both the top and center of the bilayer, indicating that cholesterol acts as a screen, preventing the lipids from direct interaction with the antibiotic. At low temperature, the ordering of the lipid in the presence of cholesterol does not change upon filipin addition, whereas at elevated temperatures the local ordering of both the lipid and the labile cholesterol is significantly lower than that in the absence of the drug. There is a very important difference between the degree of local ordering as measured by the lipids and by cholesterol at high temperatures. The orientation of cholesterol in the labile site is perpendicular to the bilayer plane. Although the orientation of cholesterol in the immobilized site cannot be calculated, it seems unlikely that a configuration of the complex in which filipin and cholesterol are parallel to the membrane surface [de Kruijff, B., and Demel, R.A. (1974)] would favor the sterol exchange between the 2 sites. The interaction between filipin and cholesterol occurs on a time scale greater than 10-5 s; this should be related to the previous 2H NMR observation that amphotericin B interacts with cholesterol on a time scale lower than 10-5s [i.e., near the nanosecond scale; Dufourc, E. J., Smith, I. C. P., and Jarrell, H. C. (1984)].