Exp Neurobiol 2019; 28(6): 658-669
Published online December 31, 2019
© The Korean Society for Brain and Neural Sciences
Min Ho Tak1, Yongwoo Jang2, Woo Sung Son3, Young Duk Yang3* and Uhtaek Oh1,4*
1Interdisciplinary Program in Neuroscience, Seoul National University, Seoul 08826, 2Department of Biomedical Engineering, Hanyang University, Seoul 04763, 3College of Pharmacy, CHA University, Seongnam 13488, 4Sensory Research Center, Brain Science Institute, Korea Institute of Science & Technology (KIST), Seoul 02792, Korea
Correspondence to: *To whom correspondence should be addressed.
Young Duk Yang, TEL: 82-31-881-7170, FAX: 82-31-881-7219
Uhtaek Oh, TEL: 82-2-958-7031, FAX: 82-2-958-7034
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Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca2+ 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 Ca2+ binding. Because the Ca2+ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca2+ 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 Ca2+ 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 Ca2+-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 Ca2+ 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 Ca2+. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca2+ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca2+ and modulate the activation by Ca2+ and voltage.