Various Physiological Factors for Regulating Activation Speeds in the Delayed Calcium-Dependent Activation of ANO6/TMEM16F

Anoctamin6/TMEM16F (ANO6) is a transmembrane protein that functions as a Ca 2+ ‐activated lipid scramblase and Ca 2+ ‐activated ion channel. Unlike other ion channels in its family, such as ANO1/TMEM16A or ANO2/TMEM16B, ANO6 shows unique characteristics of delayed ion channel activation in the presence of relatively high intracellular Ca 2+ . This property raises questions regarding its physiological function as an ion channel. Here, we demonstrate that the delayed activation of ANO6 ion channel activity can be modified by different splicing variant types and various intracellular physiological factors, including temperature, Mg‐ATP, and actin cytoskeleton. Human ANO6 has three splicing variants, designated 1, 2, and 5, with variant 1 showing the longest delayed activation, and variants 2 and 5 showing a relatively short delay in whole‐cell patch‐clamp experiments. Conversely, murine ANO6 has only one splicing variant which corresponds to human variant 1 ANO6, thus exhibiting long‐delayed activation. Using a human whole transcriptome database (e.g., TCGA, ISOexpress), we analyzed mRNA levels of each ANO6 splicing variant in human tissues. Interestingly, variant 5 was predominant in all healthy human tissues. This finding implies profound experimental and clinical importance, given that almost all ANO6 studies are performed using murine ANO6. Furthermore, the present study focused on elucidating the different activation mechanisms of ANO6 splicing variants. Similar to variants 2 and 5, variant 1 showed no delayed activation on the excised inside‐out patch‐clamp experiment, implying the existence of unknown cytosolic factors that modulate and delay the activation of ANO6 variant 1. We also discovered that increasing intracellular ATP and stabilizing the actin cytoskeleton extended delayed activation of ANO6 variant 1. Temperature also participates in ANO6 activation. Increasing the temperature to 37□ markedly reduced the delayed activation and increased the Ca 2+ sensitivity to physiological levels. Specific mechanisms through which ATP, actin cytoskeleton, and temperature modulate delayed activation remains to be studied further. Collectively, the data obtained in the present study revealed that humans and mice possess different ANO6 splicing variants with different delayed activation kinetics, and many cytosolic factors modulate delayed activation of ANO6 variant 1 which corresponds to murine ANO6. This finding is important because most studies on ANO6 utilize murine ANO6. We posit that physiological and clinical research data should be considered separately when evaluating human and murine ANO6. Support or Funding Information This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (MIST) of the Korean government (No. NRF‐2015R1C1A1A02037738).