Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain

Figures

• 

  Fig. 1. Identification of the sequence corresponding to the EF hand-like region in ANO6. (A) Amino acid sequence alignment of human ANO1 and human ANO6 sequences. The EF hand-like region in ANO1 is indicated in the red box and the corresponding sequence in ANO6 is indicated in the blue box. The green box indicates the α0a helix. The ANO6-1-6 chimera was generated based on the sequence alignment. (B) Cryo-EM structures of human ANO1 (based on PDB ID 5OYG) and human ANO6 (based on PDB ID 6QPB). Ca2+ (green sphere), the EF hand-like region in ANO1 (red loop), and the corresponding sequence in ANO6 (blue loop) are indicated. (C) Electrostatic surface potential of the EF hand-like region in ANO1 and the corresponding sequence in ANO6. (D) Overlaid structures of ANO1 (red loop) and ANO6 (blue loop). Acidic and basic amino acids are indicated.

• 

  Fig. 2. ANO6-1-6 shows higher Ca2+ sensitivity than ANO6. (A and B) Current traces of ANO6 and ANO6-1-6 in the whole-cell patch clamp. The holding voltage was 0 mV and ramp-like pulses from –100 to +100 mV were applied every 20 s with a 3-s duration. The free Ca2+ concentrations were 0 μM and 10 μM for ANO6 and ANO6-1-6. (C) Maximal current comparison of ANO6 and ANO6-1-6 currents in the whole-cell patch clamp (n = 5 for ANO6 at 0 μM Ca2+, n = 8 for ANO6 at 10 μM Ca2+, n = 4 for ANO6-1-6 at 0 μM Ca2+, and n = 5 for ANO6-1-6 at 10 μM Ca2+). (D) Time-to-peak comparison of ANO6 and ANO6-1-6 currents in the whole-cell patch clamp (n = 7 for ANO6 at 10 μM Ca2+ and n = 5 for ANO6-1-6 at 10 μM Ca2). (E) Comparison of ANO6 and ANO6-1-6 channel inactivation by normalized currents in the whole-cell patch clamp (n = 5 for ANO6 at 10 μM Ca2+ and n = 4 for ANO6-1-6 at 10 μM Ca2+). (F and G) Current traces of ANO6 and ANO6-1-6 in the inside-out patch clamp. The holding voltage was +80 mV and applied free Ca2+ concentrations are indicated. Gray dotted lines indicate 0 pA. (H) Comparison of Ca2+ sensitivity of ANO6 and ANO6-1-6 in the inside-out patch clamp (n = 4 for ANO6 and n = 4 for ANO6-1-6). The…

• 

  Fig. 3. ANO6 and ANO6-1-6 show no difference in voltage dependence. (A) Current traces of ANO6 and ANO6-1-6 in the inside-out patch clamp. The free Ca2+ concentrations are indicated above each graph. The step pulse was administered from –60 to +200 mV with +20-mV increments and was held at –60 mV. Gray dotted lines indicate 0 pA. In magnified images, gray lines are the fitted single exponential curves. (B) Current traces of ANO6 and ANO6-1-6 in the inside-out patch clamp. Initially, a +100-mV pulse was applied to activate the channel at a specific Ca2+ concentration, and voltage was applied from –100 to +100 mV with +20-mV increments and was held at –60 mV. Gray dotted lines indicate 0 pA. In magnified images, gray lines are the fitted single exponential curves. (C) Comparison of voltage-dependent conductance of ANO6 and ANO6-1-6 currents. Channel conductance was assessed from the magnitude of tail currents from (A). After normalizing to the conductance at +200 mV, the Boltzmann equation was used to calculate V50 (n = 5 for ANO6 at 11.5 μM Ca2+, n = 8 for ANO6 at 30 μM Ca2+, n = 8 for ANO6-1-6 at 6.5 μM Ca2+, and n = 5 for ANO6-1-6 at 30 μM Ca2+). (D) Activation time constant analysis of…

• 

  Fig. 4. Molecular dynamics simulations of Nt-CaRes movement. (A) Snapshots of ANO6 at 0, 300, and 1,000 ps from the molecular dynamics simulation. Amino acid letters indicate relative acidic amino acid positions in the “putative” Ca2+-binding reservoir in ANO6, which is 1DCKFRRQSEDPSCPNE16. The putative Ca2+-binding reservoir in ANO6 (black loop), Ca2+ (green sphere), simulation time, and the number of interacting Ca2+ molecules are indicated. No interacting Ca2+ was noted. (B) Time-distance graph of ANO6 simulation. Distances represent the distance between the oxygen atom in the acidic amino acid to the nearest Ca2+. The stable interaction length was calculated as 2.5 Å (gray dotted line). (C) Snapshots of ANO6-1-6 at 0, 300, and 1,000 ps from the molecular dynamics simulation. Amino acid letters indicate relative acidic amino acid positions in the putative Ca2+-binding reservoir in ANO6-1-6, which is 1DGDYNGENVEFND13. The Ca2+-binding reservoir in ANO6-1-6 (blue loop), Ca2+ (green sphere), simulation time, and the number of interacting Ca2+ ions are indicated. At 300 ps, D1 was shown to interact with Ca2+. At 1,000 ps, D1, D3, and E10 demonstrated interactions. (D) Time-distance graph…

• 

  Fig. 5. Mutations in Nt-CaRes in ANO6 decrease Ca2+ sensitivity. (A-D) Current traces of ANO6 mutations in the inside-out patch clamp. (A) E257A mutation, (B) E264A mutation, (C) D249A_E257A_D258A_E264A mutation (4A), and (D) K251A_R253A_R254A mutation (3A). The holding voltage was +80 mV and the applied free Ca2+ concentrations are indicated. Gray dotted lines indicate 0 pA. (D) Comparison of Ca2+ sensitivity of ANO6, ANO6-1-6, and the E257A, E264A, 4A, and 3A mutants in the inside-out patch clamp (n = 6 for E257A, n = 5 for E264A, n = 6 for 4A, and n = 6 for 3A). The Hill equation was used to calculate EC50. All values in (E) are expressed as mean ± SEM.

• 

  Fig. 6. Proposed model for Nt-CaRes in ANO6 and ANO1 channel activation. ANO6 or ANO1 (two orange boxes indicate a single subunit), Ca2+-binding site in transmembrane domains (empty circles in ANO6 and ANO1), Nt-CaRes in ANO6 (gray circles), Nt-CaRes in ANO1 (light blue circles), and Ca2+ (green circles) are indicated. Green arrows indicate the movement of Ca2+ to the Ca2+-binding site. ANO6 and ANO1 pores colored in red indicate a closed channel, while blue indicates an open channel. The yellow arrows indicate ion movements in the open channel.