Anion and cation permeabilities of the mouse TMEM16F/ANO6 calcium-activated channel

TMEM16F/ANO6 is widely expressed in different tissues where it plays important physiological roles, such as the regulation of blood coagulation, bone mineralization and apoptosis. TMEM16F plays two different biological roles in cells: it acts both as scramblase and as ion channel. Both of these functions are activated by the increase of intracellular Ca2+ concentration. In physiological conditions the distribution of cellular membrane lipids is polarized, meaning that the composition of the outer leaflet is different than that of the inner one. When the intracellular Ca2+ increases, the scramblases are activated. Scramblases are integral membrane proteins responsible for the translocation of phospholipids between the two leaflets of a lipid bilayer upon their electrochemical gradient, abolishing the asymmetry of the bilayer. This is a key process in many physiological and pathological contests because the exposure of particular lipids at the cell surface induces several transduction signalling cascades. Many studies investigated in details the molecular mechanisms of TMEM16F lipid scrambling and identified a specific domain of the protein which is essential for the lipid transport. The mechanisms of TMEM16F ion channel activity are less clear and, in addition, several studies reported conflicting results about the ionic selectivity of the TMEM16F-mediated current. These data were partially obtained using different methodologies and analysis making difficult to reconcile the discrepancies. In particular, studies reporting that TMEM16F is more permeable for anions were performed only using the whole-cell recordings, a technique that does not allow a direct comparison of the results with or without intracellular calcium. In contrast, results showing a higher permeability for cations derive from recordings from excised patches in the inside-out configuration. Here, we decided to investigate the ionic selectivity of TMEM16F using both configurations of the patch clamp technique, whole-cell and inside-out excised patch, using the same solutions, in order to directly compare the results. Moreover, we investigated the properties of the Q559K mutant that has been reported to alter the ionic selectivity of TMEM16F in inside-out patches. First of all, our study revealed a characteristic which is shared between the configurations. Indeed, TMEM16F heterologous expression in HEK 293T cells generates a large outward rectifying Ca2+-activated current both in whole-cell and inside-out configuration which is due to the voltage-dependent gating of the channel. On the other hand, we found consistent differences in terms of channel time-dependent activation and selectivity. In fact, although in inside-out experiments the Ca2+-dependent activation is fast and the TMEM16F-mediated current is activated in few ms, in whole-cell recordings a full activation requires up to 4 minutes. Similar results are obtained also with Q559K mutant. In inside-out patches the Ca2+ concentration for half-maximal current activation (K1/2) is 42 µM at +60 mV, while the Hill coefficient is >2. Q559K mutant shows a significant reduction of Ca2+ sensitivity with a K1/2 almost 5-folds higher and a reduction of Hill coefficient to 1.4, indicating a possible alteration of the gating mechanism. To determine the channel selectivity, we decided to calculate the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method both in whole-cell and inside-out configuration. We found that TMEM16F-mediated current is highly non-selective but there are differences depending on the configuration of the recordings. Indeed, in whole-cell both TMEM16F wild type and Q559K mutant have a PNa/PCl around 0.5 indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the Q559K mutant retains a higher permeability for Cl¯, while TMEM16F wild type channel shows a higher permeability for Na+ with a PNa/PCl reaching 3.6. These results have two major implications: First, the discrepancies found between the recordings in whole-cell and inside-out, could suggest the presence of a cellular mechanism factor which controls TMEM16F selectivity. When the patch is excised this cellular mechanism and its controlling activity are lost. Second, the residue in position 559 could be responsible to control the selectivity among cations and anions when this cellular mechanism is lost.