EF-hand like Region in the N-terminus of Anoctamin 1 Modulates Channel Activity by Ca(2+) 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.