Identification of Residues Important for Ion and Lipid Transport in a TMEM16 Scramblase

Abstract

The TMEM16 family comprises Ca2+-activated Cl- channels and phospholipid scramblases. The recent crystal structure of a fungal homologue, nhTMEM16, revealed an important architectural feature of this protein family in the form of a bilayer-spanning hydrophilic groove that is directly exposed to the membrane and provides a possible pathway for lipid translocation. Mutations that alter ion channel activity of the TMEM16 proteins localize around the groove, suggesting that the ion and lipid pathways coincide. However, an initial report suggested that nhTMEM16, unlike other TMEM16 scramblases, does not mediate ion transport. Here, we show that nhTMEM16 mediates simultaneous ion and lipid transport and that its properties closely resemble those of afTMEM16.To test the hypothesis the hydrophilic groove forms a shared pathway for ions and lipids we mutated residues facing the groove to tryptophan (Trp), as its bulky and hydrophobic side chain should impair transport. We then investigated how these mutations affect scrambling and ion transport. Of the 17 Trp-mutants tested, 40% had little to no effect on scrambling ( 50% reduction). Interestingly, the impact on ion transport closely mirrored that on scrambling, consistent with the idea that these two functions are tightly coupled. We identify a set of 3 residues which define a constriction in the groove, which when mutated to Trp cause a dramatic reduction in the rate of scrambling. Unexpectedly however, this effect is independent of the size of the introduced side chain as mutations to Alanine (Ala) has a comparably adverse impact on scrambling. Taken together our results indicate that the specific chemico-physical properties of these 3 residues lining the groove is crucial to lipid and ion transport of nhTMEM16.