Cholesterol Stabilizes Ion Channel Function during Sphingomyelinase Hydrolysis

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Sphingomyelinase, a central enzyme of sphingolipid metabolism, hydrolyses sphingomyelin to ceramide, which is a potent modulator of membrane properties. using well-defined mixtures of palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), ceramide (Cer), and cholesterol (Chol), we mimicked sphingomyelinase activity by gradually replacing sphingomyelin by ceramide. It is well-known that ceramide induces in such systems a phase coexistence of Lα and Lβ domains, where SM/Cer pairs preferentially locate in the gel domains and POPC and Chol enrich in the fluid domains. Our interests were focused on the effects of SM depletion from and Chol enrichment in the fluid domains, respectively, and in particular on the effects on ion channel function. By applying a combination of x-ray diffraction with osmotic stress we were able to determine the bending rigidities for the coexisting domains, as well as estimates for the spontaneous curvatures and Gaussian moduli of curvature. We found a small (2%) changes in bending rigidity, and curvature modulus, but a significant (46%) drop in spontaneous curvature. In the absence of cholesterol this drop is somewhat smaller (38%). However, it is way excelled by the 73% changes in bending rigidity and Gaussian curvature. using a simple hourglass-shaped model for ion channels, we calculated the resulting changes in its conformational equilibrium due to a coupling to domain elasticity. In the absence of cholesterol, we found a significant shift toward open channel states, while changes were only modest for the cholesterol containing fluid domains. This indicates a protective role of cholesterol in cellular signaling during sphingomyelinase activity.