Mechanisms of Ion and Lipid Transport by TMEM16 Scramblases

Abstract

Phospholipid scramblases collapse the plasma membrane lipid asymmetry, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Several groups showed that some TMEM16 homologues are Ca2+-dependent ion channels and/or phospholipid scramblases. The recent crystal structure of nhTMEM16 showed a possible location for the lipid pathway as each monomer exposes a hydrophilic groove (∼10 Å) to the membrane core. It was thus proposed that that during scrambling the lipid headgroups interact with the groove while the acyl chains remain in contact with the membrane's core and that ions and lipids share a common pathway. To test these hypotheses we investigated how mutants located in the groove affect scrambling and ion transport and how increasing the headgroup size of the scrambled lipids affects transport. We identified several residues lining the groove that are important for scrambling but have minimal effects on ion transport, suggesting that the two functions are not tightly coupled. Interestingly, the identified residues primarily affect scrambling in the absence of Ca2+ with lesser effects in high Ca2+, suggesting that the lipid-pathway interactions are Ca2+-dependent. We then generated NBD-labelled phospholipids with headgroups conjugated to PEG molecules as large as 5 kDa, with diameters up to 40 Å. We found that in the presence of Ca2+ all tested lipids are scrambled, despite having headgroups several fold larger than the groove. In contrast, in 0 Ca2+ lipids conjugated to PEG 2 kDa are poorly scrambled and those conjugated to PEG 5 kDa are excluded. Taken together our results suggest that the lipid pathway is narrower than in the Ca2+ bound state and that in a membrane environment the cavity could be substantially wider than seen in the structure.