EF-hand like Region in the N-terminus of Anoctamin 1 Modulates Channel Activity by Ca2+ and Voltage

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca 2+ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca 2+ binding. Because the Ca 2+ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca 2+ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca 2+ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca 2+ -coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca 2+ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca 2+ . Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca 2+ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca 2+ and modulate the activation by Ca 2+ and voltage.