Ca2+-Dependent Ion and Lipid Transport Mediated by a Fungal TMEM16 Homologue

Abstract

Ca2+-activated Cl- channels (CaCCs) play crucial roles in human physiology, from epithelial secretion to nociception and sensory transduction. Recent work showed that two members of the TMEM16 family, TMEM16A and B, encode for CaCCs. The TMEM16 family is comprised of 10 human homologues whose malfunction has been implicated in several human diseases. Despite their physiological relevance the function of the other TMEM16s is unclear and controversial. For example, TMEM16F has been reported to be a Ca2+-dependent cation channel, three different Cl- channels and to be involved in lipid scrambling (lipid transport between membrane leaflets). These data raise the possibility that not all TMEM16 proteins are CaCCs and that some might be scramblases or regulators of scrambling activity.To differentiate between these hypotheses we expressed, purified and reconstituted several TMEM16 family members and discovered that a fungal homologue simultaneously mediates ion movement and lipid scrambling. Reconstitution in planar lipid bilayers shows a non-selective ion channel of high conductance, ∼300 pS. Both transport functions are tightly regulated by Ca2+: in its absence ion channel activity is abolished and lipid scrambling is severely diminished. The apparent Km of Ca2+ for transport is ∼400 nM, a value comparable to that of other TMEM16s. We mutated a highly conserved di-acidic motif previously shown to be important for Ca2+-regulation in TMEM16A and F. Simultaneous charge-neutralization of these residues eliminates Ca2+ dependent activation of ion and lipid transport, suggesting that a single Ca2+ site regulates both.Our results demonstrate for the first time that a member of the TMEM16 family is simultaneously a Ca2+ dependent ion channel and a Ca2+ dependent lipid scramblase. This suggests that other family members, such as TMEM16F, might also be dual function proteins thus resolving the confusion regarding their function.