Figure 3.

DHA and EPA have distinct effects on the K_app of membranes with cholesterol nanodomains. (A) Chemical structures and schematics of cholesterol, DOPC, and select FAs. (B) DOPC membranes have a reduced K_app in the presence of EPA and DHA compared to no FA when low amounts of cholesterol are present. Vesicles were constructed from lipid stocks composed of chol/DOPC 25:75 (no FA) and FA/chol/DOPC 25:25:50, n ≥ 18. (C) DHA decreases the K_app of membranes assembled with higher concentrations of cholesterol compared to EPA. Vesicles were constructed from lipid stocks containing chol/DOPC 50:50 (no FA), and FA/chol/DOPC 25:45:30. Error bars represent standard error of the mean, n ≥ 23. p-values were generated by ANOVA using Dunnett’s multiple comparisons test compared to no FA. ^∗∗∗∗p ≤ 0.0001, ^∗∗p ≤ 0.01, ^∗p ≤ 0.05, ns p > 0.5. (D) Bulk FRET measurements of SUVs were used to determine the effect of EPA and DHA on nanodomain disruption. EPA inclusion increases the average distance between FRET-labeled lipids to a greater extent than OA and DHA, suggesting enhanced cholesterol redistribution with EPA. 10 mM 50 mol% chol, 50 mol% DOPC vesicles were prepared with different concentrations of equimolar 18:1 NBD-phosphoethanolamine, triethylammonium salt and 18:1 1,2-dioleoyl-sn-glycero-3-phosphoethanoamine-N-(lissamine rhodamine B sufonyl) ammonium salt. The FRET ratio (f_don/f_acc) demonstrates energy transfer efficiency between the dyes after fatty acid addition. Relative surface area was determined by normalizing the fluorescence intensity of each sample to the fluorescence intensity in fatty-acid-free PBS (data not shown). FRET studies were conducted in 300 mOsm PBS (pH 7.4), n = 3 using independent vesicle samples.