Abstract
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 Ca(2+)-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 Ca(2+)-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.
Highlights
The transbilayer asymmetric distribution of phospholipids in cell membranes is evolutionarily conserved and essential to cellular physiology
ANO6/TMEM16F has been shown to be essential for Ca2+-dependent Phospholipid scrambling (PLS), but controversy surrounds whether ANO6 is a phospholipid scramblase or an ion channel like other ANO/TMEM16 family members
Ca2+-dependent PtdSer exposure on the outer leaflet of the plasma membrane was measured by confocal imaging of two PtdSer probes, either LactoglobulinC2 fused to Clover fluorescent protein (‘LactC2’) or Annexin-V conjugated to AlexaFluor-568 (‘Annexin-V’) (Shi et al, 2006; Hou et al, 2011; Kay and Grinstein, 2011)
Summary
The transbilayer asymmetric distribution of phospholipids in cell membranes is evolutionarily conserved and essential to cellular physiology. The outer leaflet is enriched in phosphatidylcholine and sphingomyelin and the inner cytoplasmic-facing leaflet is rich in phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) (Bretscher, 1972; Fadeel and Xue, 2009; Lhermusier et al, 2011; van Meer, 2011). This asymmetry is established by ATPdependent lipid flippases, most notably members of the P4 ATPase family (Panatala et al, 2015) and ABC transporters (Borst et al, 2000), that actively transport phospholipids to one leaflet of the membrane and are important in membrane biogenesis.
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