Detection of Pure Cholesterol Bilayer Domains in Biological Membranes Overloaded with Cholesterol: Methodology Development and its Application to Porcine Lens Membrane Studies

In the eye lens’ fiber cell membranes, which are loaded with cholesterol and dense with integral membrane proteins, four purported lipid domains are expected: bulk, boundary, and trapped lipids, as well as pure cholesterol bilayer domain (CBD). Three of these domains, namely, bulk, boundary, and trapped lipids, have been identified [1]. However, showing that the CBD is present in these intact membranes, even though it was clearly detected in lens lipid membranes prepared from total lipid extracts from fiber cell plasma membranes [2], was difficult. Here, this problem was solved using an advanced saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling approach (modified discrimination by the oxygen transport method), making it possible to detect CBDs in intact fiber cell membranes. CBDs were detected only in those intact membranes in which CBDs were also detected in the lens lipid membranes. These intact membranes include those from porcine lens nuclei but not porcine lens cortices. Lens lipid membranes prepared from porcine lens cortices of two-year-old animals were CBDs free [3], while those prepared from porcine lens nuclei contained CBDs [3]. Results obtained for intact membranes, when combined with those obtained for lens lipid membranes, contribute to the understanding of the organization of lipids in fiber cell plasma membranes of the eye lens.