Project Summary Eukaryotic cells have an asymmetric distribution of lipid species across the bilayer of their plasma membrane (PM). The rapid collapse of this asymmetry, involving equilibration of lipids across the bilayer of the PM, is a vital signaling mechanism essential for many cellular processes including myoblast fusion, blood clotting and apoptotic cell clearance. Dysfunction of this process has been linked to many human diseases including bleeding disorders, cancer and autoimmunity. Equilibration of lipid species between PM leaflets is a process reliant on elusive proteins dubbed phospholipid scramblases (PLSases), which catalyze the bidirectional movement of phospholipids across the PM in a non-selective manner. Recent evidence has identified anoctamin 6 (ANO6), a putative ion channel, as an essential component of PLS activity. ANO6 is essential for Ca2+-dependent PLS activity in a wide variety of cell types, but questions remain concerning its role as a PLSase itself or as a vital regulator of this activity. Utilizing a microscopy-based scramblase assay, we have found that transfection of HEK293 cells with ANO6 induces robust Ca2+-dependent membrane scrambling that is absent in non-transfected cells. We identified a region of ANO6 essential for lipid scrambling, we term the scramblase-associated domain (SCAD), and found that insertion of this region into ANO1 (ANO1-SCAD), an ANO6 paralog with no obvious PLS activity, confers Ca2+-depedent PLSase activity, but mutating this region in ANO6 abolished this activity. Given the small size of the SCAD (12 AA), we hypothesize that this domain facilitates protein-protein interactions essential for membrane scrambling, which highlights the merit in identifying SCAD binding partners. ANO6 exists as a homodimer in vivo. Because oligomerization is important for the function of many membrane proteins including ion channels, we hypothesize that ANO6 oligomeric structure is essential for PLS activity and ion transport. We hypothesize that anoctamin 6 plays a vital role in the organization of membrane lipids and protein oligomeric structure is essential for ANO6-dependent PLS and channel activities. In Specific Aim 1 we will identify novel interactors required for the role of ANO6 in lipid organization and ion channel activity using a quantitative mass spectrometry approach to identify ANO1, ANO6 and ANO1-SCAD binding partners and compare these interactomes to distinguish ANO6 specific interactors from common ANO family interactors. In Specific Aim 2 we will investigate the requirement of Ano6 oligomerization in ANO6-dependent PLS and ion channel activities by identifying the ANO6 dimerization domain using mutagenesis and examining how the inhibition of protein dimerization through mutagenesis and the use of dominant negatives effects channel and PLS activity. The long-term goal of this project is to elucidate the mechanism(s) underlying phospholipid scramblase activity. Understanding this process will greatly increase our understanding of cell-cell fusion and provide potential targets for treating diseases in which this process is disturbed (e.g. bleeding disorders, muscular dystrophies, autoimmunity).!

Project Narrative Rapid reorganization of plasma membrane lipid asymmetry is a vital cellular signaling mechanism. Dysfunction of this process can lead to bleeding disorders, loss of cell fusion, and has been linked to several types of cancer. These experiments are poised to increase our understanding of how this rapid redistribution of lipids at the plasma membrane takes place and could yield novel targets for improving the clearance of cancer cells, increasing muscle repair, and correcting the loss of blood coagulation in bleeding disorders.