(A) Side views of OSCA1.2 (left, red), mTMEM16A (PDB 5OYB, middle, yellow), and nhTMEM16 (PDB 4WIS, right, cyan). Calcium ions are shown as green spheres in mTMEM16A and nhTMEM16. (B) Superposition of TM4 and TM6 of the structures shown in (A). The space between TM4 and TM6 in nhTMEM16 is wider than in OSCA1.2 and mTMEM16A, consistent with the lipid scramblase function of nhTMEM16 and the ion channel functions of TMEM16A and OSCA1.2 (Schroeder et al., 2008). (C) The Ca2+ binding site of TMEM16 proteins formed by residues in the cytoplasmic half of TM6, TM7 and TM8 is not conducive for Ca2+ binding in OSCA1.2 (left, red). Note, E531 in OSCA1.2 is the sole acidic residue in this region. The Ca2+ binding sites of mTMEM16A (middle) and nhTMEM16 (right) are also shown. (D) TM6 in OSCA1.2 (left), Ca2+-bound mTMEM16A (middle, yellow) and Ca2+-bound nhTMEM16 (right) have π-helical turns (pink). In the Ca2+-free structure of mTMEM16A (middle, gray, PDB 5OYG), the π-helix collapses and the helix hinges at a glycine residue of TM6. In OSCA1.2, there is also a glycine at the π-turn in TM6a, raising the possibility of structural similarities in gating transition. The helix break in TM6 of OSCA1.2 could also impart flexibility during mechanical gating.