Identification of a lipid scrambling domain in ANO6/TMEM16F

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.

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

eLife digest

Cell membranes are made of two layers of molecules called phospholipids. The types of phospholipid molecules in the outer layer are often different from those in the inner layer. This asymmetry is an important feature of most membranes, but cells also have proteins called ‘scramblases’ that can move (or scramble) the phospholipids between the two layers. This scrambling process often marks a cell for destruction but also plays a key role in many activities throughout the body including cell–cell fusion, blood clotting, autoimmune diseases and inflammation.

Previous research revealed that a membrane protein called ANO6 is needed for some kinds of phospholipid scrambling. Other proteins that are most closely related to ANO6 are not phospholipid scramblases; instead they are channel proteins that allow ions to pass across cell membranes. ANO6 can also allow ions to flow across membranes, which raised the question: is ANO6 actually a scramblase itself, or does it control other proteins with scramblase activity?

Yu, Whitlock et al. addressed this question by engineering human cells grown in the laboratory to produce the ANO6 protein, and found that these cells had high levels of phospholipid scrambling. Next, the scrambling of phospholipids in these cells was measured while the flow of ions through ANO6 was also recorded. These experiments revealed that these two processes happened almost simultaneously. Yu, Whitlock et al. suggest that this could mean that ANO6 allows ions to leak through when it shuttles phospholipids between layers of cell membranes.

ANO1 is an ion channel that is related to ANO6 but it does not have scramblase activity. By designing and testing hybrid proteins that combined parts of ANO6 and ANO1, Yu, Whitlock et al. identified the part of ANO6 that is responsible for its scramblase activity. Furthermore, computer models of this ‘scrambling domain’ suggest that it forms an unusual groove that faces into the cell membrane, and that could help phospholipids to shuttle between the inner and outer layers of the membrane. Alternatively, this groove could interact with other proteins to regulate phospholipid scrambling; and if so, further work will be needed to identify these unknown proteins.

Finally, swapping a relatively small number of features between ANO6 and ANO1 could confer scrambling activity on ANO1. This suggests that ANO1 may itself have a special relationship to membrane phospholipids. Uncovering the nature of this relationship, if it exists, as well as understanding how ANO6 scrambles phospholipids will challenge structural biologists to generate high-resolution images of these proteins in complex with phospholipids.

DOI: http://dx.doi.org/10.7554/eLife.06901.002