title: Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion.
abstract: The process of regulated exocytosis is defined by the Ca2+-triggered fusion of two apposed membranes, enabling the release of vesicular contents. This fusion step involves a number of energetically complex steps and requires both protein and lipid membrane components. The role of cholesterol has been investigated using isolated release-ready native cortical secretory vesicles to analyze the Ca2+-triggered fusion step of exocytosis. Cholesterol is a major component of vesicle membranes and we show here that selective removal from membranes, selective sequestering within membranes, or enzymatic modification causes a significant inhibition of the extent, Ca2+ sensitivity and kinetics of fusion. Depending upon the amount incorporated, addition of exogenous cholesterol to cholesterol-depleted membranes consistently recovers the extent, but not the Ca2+ sensitivity or kinetics of fusion. Membrane components of comparable negative curvature selectively recover the ability to fuse, but are unable to recover the kinetics and Ca2+ sensitivity of vesicle fusion. This indicates at least two specific positive roles for cholesterol in the process of membrane fusion: as a local membrane organizer contributing to the efficiency of fusion, and, by virtue of its intrinsic negative curvature, as a specific molecule working in concert with protein factors to facilitate the minimal molecular machinery for fast Ca2+-triggered fusion.
authors.affiliation: Department of Physiology and Biophysics, Hotchkiss Brain Institute, University of Calgary, Health Sciences Centre, Faculty of Medicine, Calgary, Alberta, T2N 4N1, Canada.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Churchward
authors.forename: Tatiana
authors.initials: T
authors.lastname: Rogasevskaia
authors.forename: Jana
authors.initials: J
authors.lastname: Höfgen
authors.forename: Jason
authors.initials: J
authors.lastname: Bau
authors.forename: Jens R
authors.initials: JR
authors.lastname: Coorssen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002784:Cholesterol / Q000172:deficiency / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis; D005372:Filipin / Q000494:pharmacology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D010866:Natamycin / Q000494:pharmacology; D012617:Sea Urchins; D022142:Secretory Vesicles; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D008244:Lysophosphatidylcholines; D047392:beta-Cyclodextrins; D005372:Filipin; D010866:Natamycin; D002784:Cholesterol; D002789:Cholesterol Oxidase; D002118:Calcium