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Temperature Dependence of Delayed Ca2+ Activation of TMEM16F (ANO6) and Its Ca2+ Sensitivity

TMEM16F, also known as ANO6, belongs to a family of putative Ca2+‐activated Cl− channels (CaCCs). In contrast to other classic CaCCs such as TMEM16A (ANO1), the activation of ANO6 requires very high cytosolic Ca2+ concentration ([Ca2+]c > 3 μM) and strong positive membrane potentials. Moreover, even when [Ca2+]c is high, the ANO6 current appears very slowly, requiring >10 min to reach a steady‐state. These properties were confirmed in all three functioning variants (V1, 2, and 5) of ANO6. The low Ca2+‐sensitivity and slow kinetics of ANO6 suggest that it does not function as an ion channel. In the present study, using whole‐cell patch recordings, we found that the three ANO6 isoforms (V1, 2, and 5) transiently transfected into HEK293T cells were activated by lower [Ca2+]c at physiological temperature (37°C). However, V2 and V5 showed significantly higher sensitivity and were activated by 400 nM [Ca2+]c and V1 by 1 μM [Ca2+]c. Furthermore, V2 and V5 showed significantly greater shortening of the latency time of activation than V1; after preparing the whole‐cell configuration, the currents reached a maximum after 543.3 ± 72.48 s, 56.67 ± 9.55 s, and 62.5 ± 4.53 s for V1, V2, and V5, respectively. The temperature‐dependent effect was reversible. In inside‐out patch, all isoforms of ANO6 exhibited higher calcium sensitivity and channel activity at 37°C than at room temperature. V3 showed no current activity in the inside‐out configuration. These results suggest that nearly all isoforms of ANO6 can be activated at low Ca2+ concentration (≤1 μM) when conditioned at physiological temperature, although they showed different kinetics. Our results suggest that ANO6 functions as ion channel in various tissues and cells depending on the variant forms of ANO6.