EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (K_a), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane K_a has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent K_a (K_app) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane K_app as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that K_app generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the K_app of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the K_app in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane K_app changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the K_app of membranes and that EPA and DHA have differential effects on K_app when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.