HEK293 Cell-Based Assay to Measure the Lipid Scrambling Activity of TMEM16 Family Members

In recent years, the elucidation of molecular mechanisms underlying lipid scrambling has raised significant attention to its implications in various physiological processes, such as blood coagulation, viral infection, cell fusion processes, and removal of apoptotic cells. This chapter focuses on a HEK293 cell–based assay tailored to assess the lipid scrambling activity of the Ca2+-activated scramblases of the TMEM16/Anoctamin family. It relies on the capacity of Annexin-V to detect the presence of negatively charged lipids and, in particular, phosphatidylserine, on the extracellular surface of the plasma membrane. Although most TMEM16 scramblases, with the exception of TMEM16F, have an intracellular localization, some of them partially localize at the plasma membrane, allowing a direct functional characterization. Annexin-V-based scrambling assay offers a versatile platform for investigating the lipid scrambling properties of TMEM16 family members and their mutants associated with genetic diseases, facilitating the elucidation of their roles in cellular physiology and pathology.