Two helices in the third intracellular loop determine anoctamin 1 (TMEM16A) activation by calcium

Abstract

Anoctamin 1 (ANO1)/TMEM16A is a Cl− channel activated by intracellular Ca2+ mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca2+. Here, we demonstrate that two helices, “reference” and “Ca2+ sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca2+-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca2+-dependent activation because neutralization of these charges change the Ca2+ sensitivity. We now predict that the Ca2+ sensor helix attaches to the reference helix in the resting state, and as intracellular Ca2+ rises, Ca2+ acts on the sensor helix, which repels it from the reference helix. This Ca2+-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1603-2) contains supplementary material, which is available to authorized users.

A novel exon in the human Ca2+-activated Cl− channel Ano1 imparts greater sensitivity to intracellular Ca2+

“…The role of CaM in Ano1 activation is still highly debated. Some studies have suggested that CaM directly binds Ano1 and plays a role in Ano1 activation (18,35). On the other hand, other studies found no requirement for CaM in Ano1 activation by Ca 2ϩ in cells (39) and when reconstituted in purified bilayers (35) and therefore suggest that Ca 2ϩ directly activates Ano1.…”

Functional roles of glutamic acid E143 and E705 residues in the N-terminus and transmembrane domain 7 of Anoctamin 1 in calcium and noxious heat sensing

“…Amino acids 444 EEEE are important for intrinsic voltage dependence, but mutating these residues has no effect on Ca 2+ sensitivity ( 7 ). Moreover, Lee and colleagues suggested that two helices in the third intracellular loop could be structurally changed in a Ca 2+ -dependent push-and-pull fashion, like a Ca 2+ sensor ( 9 ). Because Ca 2+ is a positively-charged ion, Tien and colleagues performed systematic mutagenesis of all conserved acidic residues in ANO1 ( 10 ).…”

Recent advances in TMEM16A: Structure, function, and disease

“…Y. D. Yang et al (2008) observed that the TMEM16A current activated in HEK cells coexpressing endothelin receptor subtype A was strongly outwardly rectifying, they also observed the Ca 2+ sensitivity of TMEM16A. Lee et al (2015) discovered that mutations ( 659 KMKK/ GMGG 662 ) in the two helices of the third intracellular loop alter the Ca 2+ sensitivity. The data suggested that the two helices in the third intracellular loop are essential for Ca 2+ -dependent activation .…”