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

• Published in: Experimental neurobiology Vol. 28; no. 6; pp. 658 - 669
• Main Authors: Tak, Min Ho, Jang, Yongwoo, Son, Woo Sung, Yang, Young Duk, Oh, Uhtaek
• Format: Journal Article
• Language: English
• Published: The Korean Society for Brain and Neural Sciences 01.12.2019; 한국뇌신경과학회
• Subjects: Original; 뇌과학
• ISSN: 1226-2560; 2093-8144
• DOI: 10.5607/en.2019.28.6.658

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.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.