The Protein that Held Back the Dye: Annexin's Effect on Membrane Permeability

Abstract

The membrane is more than a barrier that protects the cell; composed of lipids and protein, the membrane is implicated in signaling, cell stability, and protein interactions. They must be able to respond to stressors that can affect these roles, and employ different membrane components for that purpose. Cholesterol is a common component to both monolayers of the eukaryotic plasma membrane, moving freely and relaying of information such as changes in lipid distribution. The annexin family of membrane-associated proteins constitutes two percent of eukaryotic proteins within the cell. Annexins interact with multiple binding partners including small molecules like Ca2+, phospholipids, and other proteins that are often involved in membrane repair. To determine how binding of annexin impacts the permeability of the membrane, carboxyfluorescein (CF) release assays were performed. CF efflux from vesicles in the presence of annexin a5 without Ca2+ showed a slight decrease compared to the control of vesicles alone; however, with the addition of both annexin and Ca2+, the signal decrease was greater. In order to observe the effects of both cholesterol and protein on permeability, CF studies were repeated on vesicles containing increasing mole fractions of cholesterol. Less CF was released from vesicles containing cholesterol, and an even greater decrease was observed with annexin and Ca2+ added. This suggests that in the presence of Ca2+, annexin works to reduce the permeability of the membrane, especially for cholesterol-containing vesicles. In previous work with isothermal titration calorimetry (ITC) we also observed a change in the Ca2+ binding parameters and stoichiometry of annexin a5 in the presence of cholesterol-containing membranes. This combined data, leads us to hypothesize that through their calcium binding ability, annexins sense the distribution of lipids and help communicate changes in the membrane environment.