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 concentrations. The holding potential was +80 mV. (E) Dose-response relationship of ANO1 (●) and of its 2A (▼) and 4A (■) EF-hand like region mutants. Plots of single-channel currents at each [Ca2+]i concentration (from 0.01 to 1,000 μM) were fitted to Hill’s equation (n=5~12). Ehold=+80 mV. ANO1, EC50=1.0 μM (Hill’s coefficient (nh)=3.7); 2A, EC50=5.8 μM (nh=0.9); 4A, EC50=41.8 μM (nh=0.6). (F) Localization of wild type ANO1 and mutants in HEK cells. GFP-tagged wild type ANO1, 4A and Δ285–297 mutants are normally observed in the plasma membrane. (G) Western blot analysis of mutant ANO1s. HEK cells transfected with wild type ANO1 were used as positive control.|@|~(^,^)~|@|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 exponential fits to tail currents recorded at −120 mV.|@|~(^,^)~|@|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.|@|~(^,^)~|@|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.|@|~(^,^)~|@|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.|@|~(^,^)~|@|Surface model of mANO1. The EF-hand like region and Ca2+ ions were colored by red and green, respectively.|@|~(^,^)~|@|Schematic representation of migration of Ca2+ ions mediated by transient Ca2+ reservoir region.
Experimental Neurobiology 2019;28:658~669 https://doi.org/10.5607/en.2019.28.6.658
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