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

Figures

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  Fig. 1. Mutation of the EF-hand like region of ANO1 alters Ca2+ sensitivity. (A) Putative Ca2+ modulation sites in ANO1 and ANO2: the EF-hand like region in the N-terminus. The sequence alignment displays similarity between EF-hand loops of calmodulin and the EF-hand like regions in ANO1 and ANO2. (B) Whole cell currents of ANO1 and EF-hand like region mutants activated by [Ca2+ ]i in HEK cells transfected with ANO1 and mutant genes. Whole-cell currents were elicited using a 10 μM Ca2+ pipette solution at a holding potential of −60 mV. Pipette and bath solutions contained 140 mM NMDG-Cl. The dashed line represents the baseline. 3A; D285A/D287A/E289A mutant, 4A; D285A/D287A/E289A/D291A (AGAYAGA) mutant. (C) Summarized whole cell currents of ANO1 and EF-hand like region mutants (n=5~16). **p<0.01 compared to ANO1 (ANOVA, Tukey’s post-hoc test). Error bars represent SEMs. 5A; D285A/D287A/E289A/D291A/E294A mutant. (D) Representative traces of single-channel currents of wild-type ANO1 and its mutants activated by various concentrations of Ca2+. Ca2+ was applied to the bath of inside-out patches isolated from HEK cells expressing ANO1 and its 2A (D285A/D287A) or 4A mutant at different…

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  Fig. 2. Mutation in the EF-hand like region alters ANO1 characteristic responses to voltage steps. (A) Slow activation of outward currents at depolarization by low [Ca2+]i and slow-decaying tail currents of ANO1 at high [Ca2+]i (left panel) were almost absent in the 4A mutant (right panel). Whole-cell currents of ANO1 and its 4A mutant were recorded using a step voltage protocol (lower panel, from −100 to 100 mV in 20 mV increments, 500 ms per step) at [Ca2+]i levels of from 0 to 10 μM for ANO1 and from 0.1 to 100 μM for 4A mutant. Tail currents were recorded at −120 mV over 150 ms. (B) Current-voltage relationships of ANO1 (left panel) and 4A mutant (right panel). Data were taken from currents at the end of each voltage step (n=5). (C) The conductance-voltage relationships of tail currents (n=5). Conductances were determined by measuring instantaneous tail currents at −120 mV after various voltage pulses. (D) The upper panel displays average tail currents of ANO1 (left panel) and 4A mutant (right panel) (n=5~6). The lower panel displays deactivation time constants obtained from the tail currents shown in the upper panel. Deactivation time constants (τd) were obtained using single…

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  Fig. 3. Mutation of the EF-hand like region of ANO2 confers Ca2+ sensitivity. (A) The sequence alignments of EF-hand like regions in the ANO family. ANO1 and ANO2 have consensus sites in the EF-hand like region. Red; negatively-charged, pink; polar uncharged, green; positively charged, and blue; hydrophobic amino acid side chain. (B) Dose-response relationships of ANO1 (red lines) and ANO2 (black lines) activation by various concentrations of Ca2+ applied to the baths of inside-out patches of ANO1 or ANO2-transfected HEK cells (n=5~8) at Ehold −80 mV (straight line) or +80 mV (broken line). ANO1 at +80 mV, EC50=0.8 μM (nh=2.4), ANO1 at −80 mV, EC50=1.4 μM (nh=3.4), ANO2 at +80 mV, EC50=2.6 μM (nh=10.4), ANO2 at −80 mV, EC50=12.5 μM (nh=4.9). (C) Whole-cell currents of ANO2 (upper panel) and of ANO2 D316A/E318A/D320A mutant (lower panel) recorded from HEK cells transfected with ANO2 or its mutant with various [Ca2+]i (from 1 to 300 μM) in the pipette solution. (D) The dose-response characteristics of the whole-cell currents of ANO1 (●), ANO2 (■), and ANO2 D316A/E318A/D320A mutant (▲) (n=5~10). Ehold=−60 mV. ANO1 EC50=1.6 μM, ANO2 EC50=20.0 μM, ANO2 D316A/E318A/D320A mutant EC50=53.4 μM.

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  Fig. 4. Three-dimensional structures of ANO1. (A) The secondary structure of ANO1 was shown with ribbon presentation method, colored by yellow (left subunit of dimer), cyan (right subunit of dimer), green (Ca2+ ions), and red (EF-hand like region, 285~297 residue numbers), respectively. (B) View of dimer structure of ANO1 from extracellular side. (C) View of structure from the membrane looking towards EF-hand like region (colored by red) and Ca2+ binding site (shown with greed colored Ca2+ ions), respectively. (D) Close-up view of EF-hand like region and Ca2+ binding site.

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  Fig. 5. Comparison of three-dimensional structures of ANO1. (A) Modeled structure of mANO1 (cyan colored), structure from cryoEM (gold colored), and structure from x-ray crystallography (magenta colored) were superimposed using UCSF Chimera Match Maker module. (B) View of structures of ANO1 from extracellular side.

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  Fig. 6. Surface model of mANO1. The EF-hand like region and Ca2+ ions were colored by red and green, respectively.

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  Fig. 7. Schematic representation of migration of Ca2+ ions mediated by transient Ca2+ reservoir region.