TMEM16 Proteins: Membrane Channels with Unusual Pores

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TMEM16 proteins, also known as anoctamins, appear as a very intriguing class of biological macromolecules. The study by Lee et al. (1) adds another piece of evidence indicating the possibility that some anoctamins are both lipid scramblases and ion channels with unusual properties.

TMEM16A and TMEM16B (anoctamins 1 and 2) were initially found as membrane proteins working as Ca²⁺-activated Cl⁻ channels (2, 3, 4). It was presumed that the other eight members of the anoctamin family are also Cl⁻ channels. The big surprise came when TMEM16F was identified as a Ca²⁺-activated lipid scramblase (5). Scramblases favor the translocation of phospholipids from one to the other leaflet of cell membranes. In particular, TMEM16F appears particularly important in the exposure of phosphatidylserine on the cell surface (5), a phenomenon that occurs in a variety of physiological and pathological processes (6).

Recent studies have indicated that TMEM16F and other anoctamins have dual activity. First, induced expression of TMEM16F in cells generates both lipid scrambling and ion transport (7, 8), although the latter activity is characterized by poor discrimination between anions and cations. In contrast, TMEM16A and TMEM16B have a relatively high selectivity for anions (2, 3, 4). Second, introduction of specific mutations into TMEM16F primary sequence affects both activities (8). Third, and finally, reconstitution of a purified fungal TMEM16 protein (afTMEM16) into artificial membranes results in the appearance of lipid scrambling and nonselective ion transport, both activated similarly by Ca²⁺ (9). All these findings suggest that the translocation of lipids and the transmembrane ion flux are directly mediated by the same protein. A possible explanation for this dual function was provided when the structure of another fungal TMEM16 protein (nhTMEM16) was determined by x-ray crystallography (10). A groove on the side of nhTMEM16 was detected. This groove could provide the hydrophilic environment required to allow translocation of phospholipid polar heads. However, the study on nhTMEM16 confirmed scramblase function but did not detect ion transport (10) despite using similar methods. The study by Lee et al. (1) reveals that the lack of ion transport in nhTMEM16 could depend on the experimental conditions. In particular, the lipid composition of the artificial membrane was found to strongly affect nhTMEM16 and afTMEM16 functions. In the absence of egg phosphatidylcholine, ion transport was abolished whereas lipid scrambling was only partially affected (1). These results indicate that the lipid environment affects TMEM16 protein function, possibly also in more physiological situations.

In conclusion, it can be argued that during evolution TMEM16 proteins were initially molecules with a major function as Ca²⁺-dependent scramblases. Possibly, ion transport was a byproduct of phospholipid translocation. Intriguingly, and in contrast to what happens in more canonical channels, ion flux in TMEM16 scramblases would occur through a pore that is not entirely formed by the protein but is in part lined by lipids. Later in evolution, some anoctamins have lost lipid scrambling function and have acquired a clearer role as ion-selective channels.

Several aspects of TMEM16 proteins remain to be elucidated. In particular, it is unknown if anoctamins with pure ion channel function, such as TMEM16A and TMEM16B, have hybrid lipoproteic pores. If so, we would expect that channel properties, including conductance and selectivity, are dynamically regulated by membrane lipid composition. On the other hand, for TMEM16 proteins with dual function, we do not know if lipid translocation and ion flux occur simultaneously or depend on two separate conformational states of the protein. Future studies, possibly involving protein purification and reconstitution as in the study by Lee et al. (1), will help to answer these questions.

Editor: Michael Pusch.