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

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca activated chloride channel. Recently, the structure determination of a fungal 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 binding. Because the Ca binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca 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 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-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 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. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca and modulate the activation by Ca and voltage.