Exp Neurobiol.  2019 Dec;28(6):658-669. 10.5607/en.2019.28.6.658.

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

• Tak MH
• Jang Y
• Son WS
• Yang YD
• Oh U

• KMID: 2468883
• DOI: http://doi.org/10.5607/en.2019.28.6.658

Abstract

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca²âº 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 Ca²âº binding. Because the Ca²âº binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca²âº 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 Ca²âº 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 Ca²âº-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 Ca²âº 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 Ca²âº. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca²âº transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca²âº and modulate the activation by Ca²âº and voltage.

Keyword

Anoctamin-1; Chloride channels; Calcium; Mutagenesis; Site-directed

MeSH Terms

Amino Acids, Acidic
Animals
Calcium
Calmodulin
Chloride Channels
Cryoelectron Microscopy
Crystallography, X-Ray
EF Hand Motifs
Mice
Models, Molecular
Mutagenesis
Nectria
Protein Isoforms
Amino Acids, Acidic
Calcium
Calmodulin
Chloride Channels
Protein Isoforms