Sizing up the Lipid Pathway in a TMEM16 Phospholipid Scramblase

Abstract

Phospholipid scramblases collapse the plasma membrane lipid asymmetry, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family were shown to function as Ca2+-dependent phospholipid scramblases as well as ion channels in cells and in reconstituted systems. The recent crystal structure of nhTMEM16 identified a hydrophilic groove, ∼11 A wide, exposed to the hydrophobic core of the membrane as the putative lipid pathway. It was proposed that lipid scrambling occurs via phospholipid headgroup-groove interactions. To test this mechanism and to gain insights into the properties of the lipid pathway we investigated whether there is a headgroup size cut-off. We generated NBD-labelled phospholipids with PEG molecules of increasing size (PEG1000-5000) attached to the headgroup and monitored scrambling by afTMEM16. We found that increasing the headgroup size up to PEG2000 (diameter ∼40 A) does not alter scrambling in high Ca2+, and even lipids conjugated to PEG5000 (diameter ∼50 A) are scrambled albeit less well. This suggests that the cut off is considerably larger than the width of the cavity. In the absence of Ca2+ TMEM16 scramblases show substantial activity. We found that the selectivity of the apo state differs from that of the bound state as they have different headgroup size selectivity: lipids conjugated with PEG2000 are poorly scrambled and PEG5000 is completely excluded. This is consistent with the idea that the activity in the apo state reflects an incomplete closure of the lipid pathway, which is narrower than in the Ca2+ bound state by ∼10 A. Taken together these results suggest that interactions between the groove and the lipid headgroup do not play a key role in scrambling or, alternatively, that in a membrane environment the cavity could be substantially wider than in the structure.