Phospholipid scrambling (PLS) is a ubiquitous cellular mechanism involving the regulated bidirectional transport of phospholipids down their concentration gradient between membrane leaflets. ANO6/TMEM16F has been shown to be essential for Ca²⁺-dependent PLS, but controversy surrounds whether ANO6 is a phospholipid scramblase or an ion channel like other ANO/TMEM16 family members. Combining patch clamp recording with measurement of PLS, we show that ANO6 elicits robust Ca²⁺-dependent PLS coinciding with ionic currents that are explained by ionic leak during phospholipid translocation. By analyzing ANO1-ANO6 chimeric proteins, we identify a domain in ANO6 necessary for PLS and sufficient to confer this function on ANO1, which normally does not scramble. Homology modeling shows that the scramblase domain forms an unusual hydrophilic cleft that faces the lipid bilayer and may function to facilitate translocation of phospholipid between membrane leaflets. These findings provide a mechanistic framework for understanding PLS and how ANO6 functions in this process.

DOI: http://dx.doi.org/10.7554/eLife.06901.001