Endogenous Ca²⁺-activated chloride channels (CaCCs) regulate anionic currents by intracellular Ca²⁺ as well as by voltage [1–4]. Although CaCCs mediate diverse functions in various tissues, their biophysical properties display remarkable homogeneity. Their typical properties can be summarized as; activation by intracellular Ca²⁺ and voltage, an outwardly rectifying current-voltage relationship, slow time-dependent activation by voltage pulses, and voltage-dependent slow decay of tail currents [2]. Anoctamin 1 (ANO1) cloned for a CaCC has the biophysical and the pharmacological properties of native CaCCs [5–7]. For example, it is activated by Ca²⁺- and voltage, blocked by CaCC inhibitors, stimulated by G-protein-coupled receptor ligands via the PLC/IP₃ pathway, expressed in transport epithelia, and has an outwardly rectifying I~V relationship [5–7]. ANO1 has four different splice variants, and of these, splice variants with exon 6b or exon 13 are known to affect Ca²⁺ sensitivity [8].

Because CaCCs are essential for mediating various physiological functions, an understanding of their activation mechanisms induced by intracellular Ca²⁺ and voltage would provide important insight of their functions. Several studies proposed the gating mechanism and putative binding residues by intracellular Ca²⁺. For example, Xiao and colleagues report that a poly-glutamate region in the first intracellular loop is important for coupling the voltage- and Ca²⁺-dependent gating of ANO1 [9]. Moreover, Lee and colleagues suggested that two helices in the third intracellular loop coordinate a conformational change in a Ca²⁺-dependent push-and-pull fashion [10]. Based on a positively charged ion of Ca²⁺, Tien and colleagues conducted systemic mutagenesis of all conserved acidic residues of ANO1, and concluded four putative amino acids (E650, E698, E701, and E730) are critical for Ca²⁺-induced ANO1 activation [11]. Recently, the crystal structure of the fungus Nectria haematococca TMEM16 (nhTMEM16) has been identified [12]. Based on the crystal structure, a calcium binding site has been proposed. The nhTMEM16 structure reveals that a putative Ca²⁺ binding site within subunit cavity is positioned by acidic amino acid residues in alpha-helices 6 and 7. The Ca²⁺-dependent lipid scrambling is disappeared by point mutations on several residues on glutamate and aspartate. More recently, mouse ANO1’s structure further characterized by cryo-electron microscopy (cryo-EM) in 3~4 A resolution, which turned out a hydrophilic membrane-exposed cavity that is a path for lipids in scramblases has changed to form an enclosed aqueous pore in a TMEM16 channel [13].

Although the Ca²⁺ binding site was determined in an nhTMEM16, the action mechanism underlying the activation of mammalian ANO1 and ANO2 by Ca²⁺ has still remained unclear. Based on the crystal structure of an nhTMEM16, the Ca²⁺ binding motif is deeply embedded inside phospholipid bilayer. Therefore, it is assumed the modulatory residues to help the movement of Ca²⁺ ions to the inside Ca²⁺ binding pocket of ANO1. In the present study, we set out to find the important residues to modulate channel activation of mouse ANO1. Especially, we investigated the role of the EF-hand like region in the N-terminus of ANO1 in both Ca²⁺- and voltage-dependent ANO1 activation.

Mutagenesis and gene expression

All mutants were generated from the wild type construct, mouse ANO1 (pEGFP-N1-mANO1). Amino-acid substitution or deletion mutants were made using a site-directed mutagenesis kit (Muta-direct, iNtRON Biotech, Seoul, Korea) or by the overlap-PCR method. Mutations in all mutants were confirmed by sequencing whole nucleotide sequences.

HEK 293T cells were transfected with 1 μg of pEGFP-N1-mANO1 or pEGFP-N1-mutants, 0.05 μg pEGFP-N1, and FuGENE (Roche Diagnostics, Penzberg, Germany) in 35-mm Petri dishes. Transfected cells were incubated in DMEM supplemented with 10% fetal bovine serum (GIBCO) and penicillin-streptomycin at 37°C in a 5% CO₂ incubator. Cells were used one or two days after plating.

Electrophysiology

As soon as a borosilicate glass pipette (World Precision Instruments, Sarasota, FL) touched the surface of a HEK cell, a gentle suction was applied to the pipette to obtain gigaseal. The tip resistance of the pipette was about 3 Mohms for whole-cell recordings. To record whole-cell currents, the membrane in contact with the pipette was ruptured by applying gentle suction after forming gigaseals. Once a whole cell was formed, the capacitive transients were canceled. Currents were recorded with a patch-clamp amplifier (Axopatch 200B, Molecular Devices) and filtered at 5 KHz. The whole-cell currents activated by each Ca²⁺ concentration (0.1~100 uM) were recorded from a holding potential of −60 mV during voltage clamp steps from −100 to+100 mV by 20 mV increments with 765 ms (−60 mV : 65 ms, −100~100 mV : 500 ms, −120 mV : 200 ms) total duration. Data from the amplifier were digitized with Digidata 1440A (Molecular Devices) and stored in a personal computer.

ANO1 currents were activated by intracellular Ca²⁺ at a holding potential of −60 mV unless specified. For recording currents in response to voltage pulses, membrane potential was changed from −100 mV to+100 mV in 20 mV increment. Square voltage pulses of 500 ms duration were delivered from a holding potential of −60 mV. Tail currents were recorded at −120 mV.

The control pipette solution contained 140 mM NMDG-Cl, 2 mM MgCl₂, and 10 mM HEPES adjusted to pH 7.2. For the Ca²⁺–free solution, 1 mM EGTA was added to the control solution.

To calculate the accurate free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in pipette solutions, the WEBMAXC program in website (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm) was used. The bath solution contained 140 mM NMDG-Cl, 2 mM MgCl₂, and 10 mM HEPES and was adjusted to pH 7.2.

Whole cell peak currents were recorded at each free [Ca²⁺]_i, and then averaged for subsequent analysis. Half-maximal concentrations (EC₅₀) were calculated using averaged peak currents, and fitted using the Hill equation. Deactivation time constants (τ_d) were derived by the single exponential fitting of tail currents recorded at −120 mV for 150 ms.

Molecular modeling of ANO1

To construct three-dimensional structure of mouse ANO1 using in silico method, the crystal structure of TMEM16 from the fungus Nectria haematococca (PDB ID:4WIS, nhTMEM16) was used [12]. Using I-TASSER [14] algorithm, the modeled structures were generated with template structure (4WIS), and energy minimization of structures were performed with the Amber force field [15, 16] with UCSF Chimera [17]. During energy minimization, AMBER force field parameters were set to the basic values in UCSF Chimera and Antechamber module was used to adjust parameters to non-standard residues. In energy minimization step, steepest descent (100 steps) and conjugate gradient (100 steps) minimization were used to relieve unfavorable clashed region. Size of all minimization steps was 0.02 Å. Structural alignment of modeled structure and nhTMEM16 was performed using MatchMaker modules in UCSF Chimera. During structural alignment, Needleman-Wunsch alignment algorithm was used with Blosum-62 matrix and 30% secondary structure score [18]. Structural matching process was iterated by pruning long atom pairs until no pair exceeds of 2.0 angstroms. For structure refinement analysis in water solvent, the adaptive Poisson-Boltzmann software (APBS) [19] and Coulombic surface coloring method in UCSF Chimera were used to analyze the electrostatic surface potential (ESP) of modeled structures. The electrostatic surface potential maps of structures were generated by numerically solving the Poisson-Boltzmann equation based on molecular mechanics. Structure validation was performed with Protein Structure Validation Software Suite (PSVS) [20]. Molecular visualization of structure was done using UCSF Chimera. Also, Probis algorithm was used to predict potent Ca²⁺ modulating site of ANO1 [21].

Mutation in the EF-hand like region of ANO1 changes activation kinetics evoked by Ca²⁺

In mammals, calmodulin is a multifunctional intermediate protein that is regulated by Ca²⁺. The intracellular Ca²⁺ ions have well known to directly bind to EF-hand motif that is an signature sequence of the most common (canonical) EF-hand (-DxDx-DGxxxxxE-) of calmodulin [22, 23]. Interestingly, we first found that the amino acid residues (285-DGDYEGDNVE-294) in the N-terminus of ANO1 are somewhat similar to the EF-hand motif of calmodulin (Fig. 1A). Thus, we designated this region as the EF-hand like region in the present study. To investigate the effect of EF-hand like region on the activity of ANO1, we constructed ANO1 mutants in the region and then compared their activity by Ca²⁺ and voltage with those of the wild-type.

After forming whole cells with 10 μM Ca²⁺ in the pipette, large, robust currents were evoked in HEK cells transfected with wildtype ANO1. However, whole-cell currents to Ca²⁺ desensitized rapidly (Fig. 1B and 1C). In contrast to wild-type ANO1, whole-cell currents of Δ285~297 mutant to 10 μM Ca²⁺ were barely detectable (Fig. 1B and 1C). Therefore, Ala substitution was performed to determine the important amino acids in this region. More specifically, negatively charged amino acids, Asp or Glu at positions 285, 287, 289, or 291 were replaced by Ala. The Ala-substitution analysis revealed that D287A and D291A mutants elicited current responses comparable to that of wild type ANO1. However, the D285A and E289A mutants showed significant reductions in current response (Fig. 1B and 1C). The double (D285A/D287A) and triple Ala (D285A/D287A/E289A, 3A) substitution mutants also showed significant reductions in current density to 10 μM Ca²⁺ (Fig. 1B and 1C). When four or five acidic amino acids in 285-DGDYEGDNVE-294 were replaced with Ala, the mutants AGAYAGA (4A) and AGAYAGANVA (5A) showed marked reductions in current response to 10 μM Ca²⁺ (Fig. 1B and 1C), suggesting that all five acidic amino acids in this region contribute to activation of ANO1 by Ca²⁺.

To further investigate the responsiveness to Ca²⁺, different concentrations of Ca²⁺ were applied to the bath of inside-out membrane patches to investigate dose-response relationships. Because single-channel currents of ANO1 mutants activated by Ca²⁺ were too small to measure at −60 mV, the holding potential was set at +80 mV in order to increase current amplitudes. When the concentration of Ca²⁺ was increased from 0.01 μM to 10 μM, macroscopic currents were observed to increase dose-dependently (Fig. 1D and 1E). The effective concentration (EC₅₀) of Ca²⁺ for the activation of wild-type ANO1 was 1.0 μM (n=5~11). Endogenous CaCCs are known to have a greater sensitivity to Ca²⁺ at depolarization [2–4]. Likewise, ANO1 also shows a slight increase in sensitivity to Ca²⁺ at depolarization. At −80 mV, the EC₅₀ of ANO1 was 1.4 μM (n=5~12). Furthermore, the macroscopic channel currents of ANO1 increased abruptly between 0.3 and 3 μM. Thus, the Hill coefficient of wild-type ANO1 was found to be 3.7, suggesting a high level of cooperativity among Ca²⁺ coordination sites. In contrast, magnitudes of macroscopic currents of AGAYAGA mutant activated by Ca²⁺ were much smaller than those of the wild-type (Fig. 1D and 1E). Furthermore, the dose-response curve of this mutant was markedly shifted rightward with a 35-fold increase in EC₅₀ (41.8 μM, n=6~16) versus wild-type ANO1 (Fig. 1D and 1E). Interestingly, the Hill-coefficient of the mutant was markedly lower at 0.6, suggesting a lack of cooperativity among Ca²⁺ coordinating sites. Similarly, when two acidic amino acids in the EF-hand like region were replaced with Ala (D285A/D287A), the EC₅₀ was 5.8 μM, which represented a much smaller rightward shift than that observed for the AGAYAGA mutant. The Hill-coefficient of the D285A/D287A mutant was 0.9, again lower than that of the wild-type ANO1. It is possible that these mutations could alter protein stability or cell surface expression, which would result in impaired channel activity. Thus, to confirm the cell surface expression and protein stability of mutants, we constructed GFP-tagged wild type ANO1 and mutant ANO1 proteins. Transfection of these plasmids into HEK cells showed normal localization to the plasma membrane and protein expression level in both wild type and mutants (Fig. 1F and 1G). These results suggest that acidic amino acids in the EF-hand like region of ANO1 are essential for the activation by Ca²⁺. Furthermore, these results also suggest that the EF-hand like region of ANO1 will serve to transfer Ca²⁺ to the Ca²⁺ binding site through multiple interactions with Ca²⁺.

Mutation in the EF-hand like region of ANO1 changes activation kinetics evoked by voltage

Voltage is also required for the activation of ANO1 [5]. However, the location in ANO1 responsible for its reaction to voltage pulses is not known. To examine ANO1 responses to voltage, voltage pulses from −100 mV to 100 mV were applied in 20 mV increments to HEK cells expressing the wild-type or AGAYAGA mutant. As was observed for endogenous CaCCs [2, 3], ANO1 was rarely activated by voltage with no Ca²⁺ in the pipette (no added Ca²⁺ and 5 mM EGTA), but it was activated by voltage pulses when the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased from 0.1 to 10 μM (Fig. 2A). Furthermore, as [Ca²⁺]_i was increased, current responses of wild-type ANO1 to voltage steps also increased (Fig. 2A). At depolarization, instantaneous ANO1 activation was followed by a slow activation that achieved peak current after a few hundred milliseconds (Fig. 2A). However, this slow activation was not observed at voltage pulses of less than 0 mV. Furthermore, the typical slow activation induced by depolarization became less time-dependent when [Ca²⁺]_i was increased. Currents activated by voltage pulses were outwardly rectifying because ANO1 elicited much greater currents at depolarization than at hyperpolarization (Fig. 2B). Thus, ANO1 conductances were greater at depolarization (Fig. 2C). Similarly, ANO1 conductance was also found to be Ca²⁺ dependent; and conductance at each voltage increased as [Ca²⁺]_i was increased from 0.1 to 10 μM (Fig. 2C).

To investigate the kinetics of deactivation, tail currents activated by −120 mV after various pre-pulses were measured at different [Ca²⁺]_i. As was observed for the tail currents of endogenous CaCCs [2, 4, 24], ANO1 tail currents decayed slowly as the pre-pulse depolarized (Fig. 2A and 2D), which suggests the voltage-dependent deactivation of ANO1. Furthermore, tail current decay was also found to be Ca²⁺ dependent, as tail currents decayed more slowly on increasing [Ca²⁺]_i (Fig. 2D). The deactivation time constant (τ_d) was well fitted by a single exponential. τ_d increased as [Ca²⁺]_i increased, especially at [Ca²⁺]_i>1 μM. But at low [Ca²⁺]_i (<0.3 μM), τ_d did not change appreciably as voltage was changed. Furthermore, hyperpolarization (−100 mV) deactivated ANO1 rapidly even in the presence of high [Ca²⁺]_i (10 μM) (Fig. 2A and 2D).

In contrast, the AGAYAGA mutant responded to voltage pulses differently from the wild-type. More specifically, this mutant showed much lower current responses to voltage pulses (Fig. 2A), and its I~V curves at different [Ca²⁺]_i were close to linear (Fig. 2B). Furthermore, the conductance of AGAYAGA was less dependent on voltage (Fig. 2C). In addition, its activation and deactivation kinetics were quite different from those of wild type ANO1. The characteristic slow activation observed at the depolarizing voltage was almost absent in the AGAYAGA mutant (Fig. 2A). Thus, its activation by voltage appeared time-independent. Similarly, unlike the wild-type, its deactivation was largely voltage independent at all [Ca²⁺]_i examined, and τ_d was almost constant throughout the voltage pulse range (Fig. 2D). Thus, the EF-hand like region of ANO1 appears to contribute substantially to its voltage dependence.

The EF-hand like region in ANO2

Of the 10 known isoforms of the anoctamin channel family, ANO1 and ANO2 are known to be activated by Ca²⁺ [5, 25]. Thus, we aligned the ~45 amino acids flanking the EF-hand like regions of all ANO homologs. As shown in Fig. 3A, acidic amino acids in 285-DGDYEGDNVEFND-297 of ANO1 were found to be well conserved in ANO2, but not in the other ANO homologs. This coincidence of homology and Ca²⁺ response also suggests that the EF-hand loop-like region is involved in the modulation for the Ca²⁺ sensitivity of ANO1 and ANO2.

ANO2 was found to be activated by [Ca²⁺]_i in a dose-dependent manner (Fig. 3B). ANO2 also showed voltage-dependent Ca²⁺ sensitivity (Fig. 3C and 3D). At a holding potential of −80 mV, the EC₅₀ for the Ca²⁺ activation of ANO2 was 12.5 μM. However, at +80 mV the EC₅₀ reduced to 2.6 μM. Therefore, as observed in ANO1, ANO2 also showed a greater sensitivity to Ca²⁺ at depolarization than at hyperpolarization.

Because single-channel currents of ANO2 mutants were too small to measure even at the maximum Ca²⁺ concentration used (300 μM), whole-cell currents of wild-type ANO2 and of its D316A/E318A/D320A mutant were used to obtain an approximation of the dose-response relationships of ANO2 mutants (E_hold=−60 mV). The overall shape of ANO2 current response to Ca²⁺ was found to be similar to that of ANO1, except that a higher [Ca²⁺]_i was required for activation. Appreciable currents were activated only by Ca²⁺ concentrations higher than 3 μM. The EC₅₀ of ANO2 was found to be 20.0 μM, which is a 20-fold greater than that of ANO1 (EC₅₀=1.0 μM) (Fig. 3C and 3D). When three acidic amino acids (316-DGEYDSPGDDMND-328) in the EF-hand like region in ANO2 were replaced with Ala (D316A/E318A/D320A), Ca²⁺ sensitivity and current density were substantially reduced (EC₅₀=54.3 μM vs 20 μM for wild type ANO2) (Fig. 3C and 3D). These acidic amino acids correspond to the 1, 3, and 5 residues of the common EF hand of calmodulin, and are known to be essential for Ca²⁺ coordination [23]. These results further suggest that the acidic amino acids in the EF-hand like regions of ANO1 and ANO2 are an important modulatory residue in Ca²⁺-induced ANO1 activation.

Structural properties of ANO1 from molecular modeling analysis

To ascertain the 3D structure-based involvement of EF-hand like region in Ca²⁺ binding to ANO1, we performed molecular modeling analysis using various in silico methods such as I-TASSER, ROSETTA [19], RaptorX [26], Phyre2 [27]. All modeled structures were validated using PSVS to select reasonable structure among predicted structures, and several modeled structures from I-TASSER algorithm was selected for further refinement. Finally, the best one structure among modeled structures was selected based on energy-scoring function and validation results. This model shows that the overall folding of ANO1 structure was similar with that of nhTMEM16 structure used as template during homology modeling (Fig. 4).

The three-dimensional structure of ANO1 shows a similar overall shape when compared to the nhTMEM16 structure used for modeling (Fig. 5). ANO1 has bundle of transmembrane helices like nhTMEM16, and specifically, the N-terminal region is longer than nhTMEM16. The long N-terminal region is composed of more than 120 amino acids and is expected to form a flexible loop rather than a transmembrane helix. Before reaching the first TM, several helix forms existed and some helical region seems to be able to bind Ca²⁺ ion. In modeled structure, the EF-hand like region was located at the entrance leading to the Ca²⁺ binding site (Fig. 6). Therefore, this EF-hand like region could have effect on the migration of Ca²⁺ ions between the cytoplasm and the Ca²⁺ binding site (Fig. 4 and 7).

Activation of ANO1 by Ca²⁺ and voltage

As is observed for native CaCCs [2, 4, 24], the rate of ANO1 activation by depolarization increases with [Ca²⁺]_i. In addition, the deactivation of ANO1 was also found to be Ca²⁺ and voltage dependent. Kuruma et al. [2] derived a model for the Ca²⁺- and voltage-dependent activation and deactivation mechanisms of native CaCCs, which fits well with actual current responses to Ca²⁺ and voltage. This model contains three main features. First, the activations of CaCCs depend mainly on Ca²⁺, and not on voltage. Second, more than one Ca²⁺ ion is required to activate CaCCs, and third, deactivation depends on voltage. Evidently, ANO1 appears to possess all three of these features of endogenous CaCCs. First, ANO1 was easily activated by Ca²⁺ but rarely activated by voltage at low [Ca²⁺]_i (Fig. 2A). Second, the Hill coefficient of Ca²⁺ for the activation of wild-type ANO1 was 3.7, and this reduced to less than 1 when Glu and Asp residues in the EF-hand like region were replaced by Ala. These results suggest that the binding of more than one Ca²⁺ is required for ANO1 activation and that acidic amino acids in the EF-hand like region cooperatively collect and transport Ca²⁺. Third, the deactivation of ANO1 is voltage dependent, because hyperpolarization was found to deactivate ANO1 even in the presence of Ca²⁺ (Fig. 2). Thus, the mechanisms underlying the activation and deactivation of ANO1 by Ca²⁺ and voltage appear to be similar to those proposed for native CaCCs.

Mechanism of Ca²⁺ binding and subsequent ion conduction of mANO1

The Ca²⁺ binding of sites of ANO1 are well characterized in detail in cryo-EM studies [13, 28]. According to these studies, Cl− conducting pores are present in each subunit, which are formed by transmembrane helices α3-α7. The pore has a narrow neck that determines Cl− conductance and wide vestibule [13, 28]. When Ca²⁺ binds four acidic amino acids in α7 and α8, the positive charge density in α7 and α8 attracts the E654 residue in α6, which makes a comparably large movement of α6. This conformational change of α6 induces the opening of the neck of the pore, thus conducting Cl⁻ [13]. Mammalian ANO1 has longer cytoplasmic N-terminal extension than nhTMEM16, whose function is not well characterized. The long region of N-terminal is located adjacent to the membrane surface rather than the region passing through the membrane. We do not know how the EF-hand like region in the N-terminus links to the Ca²⁺ binding sites in α6, α7, or α8. The strong phenotype of mutants in this region clearly suggests its role in modulating Ca²⁺-dependent activation of ANO1. Perhaps, the EF-hand like region acts like a transient Ca²⁺ reservoir, which could help the Ca²⁺ ions move to the Ca²⁺ binding site embedded inside the lipid bilayer (Fig. 7).

The EF-hand like region controls voltage dependence of ANO1

One of the canonical properties of CaCCs is its activation by Ca²⁺ and voltage [2]. As a CaCC, ANO1 is also activated by voltage and Ca²⁺ [5, 7, 9, 29]. The activation of ANO1 by voltage requires intracellular Ca²⁺ because without Ca²⁺, depolarization fails to open ANO1 [9, 29]. Thus, it seems the voltage- and Ca²⁺-dependent gating of ANO1 is tightly coupled. A few sites were suggested to be essential for the voltage- and Ca²⁺-dependent activation of ANO1. Structural analysis combined to mutagenesis study suggests that the sixth TM is essential for Ca2+ as well as voltage-dependent gating of mouse ANO1 [30]. A highly charged segment in the first intracellular loop (444–EEEEEAVK-451), an intracellular loop between TM α2 and TM α3, is important for Ca²⁺- and voltage-dependent gating [9, 29]. Deletion or mutation of EAVK residues profoundly reduced Ca²⁺ and voltage-activated currents suggesting the Ca²⁺ and voltage sensitivities are also coupled [9, 29]. The EF-hand like region in ANO1 appears to control the voltage dependence of ANO1. When we replaced four acidic amino acids in the EF-hand like region with Ala (the AGAYAGA mutant), the slow activation of ANO1 by voltage at low Ca²⁺, voltage-dependent conductance change, and voltage dependent increases in τ_d were largely eliminated (Fig. 2), which strongly suggest that the EF-hand like region also controls the voltage dependence of ANO1. As the voltage dependency relies on the binding of Ca²⁺ to the Ca²⁺ binding sites in the TM α6 whose conformational change gates the channel, it is conceivable that the ionic interaction between Ca²⁺ and oxygen atoms of carboxyl or carbonyl groups of the acidic amino acids in the EF-hand like region function as a voltage sensor. Voltage change in this region may affect the ionic interaction between Ca²⁺ and carboxyl or carbonyl groups in the region. Alternatively, the acidic amino acids in the EF-hand like region may interact with voltage sensors in the pore region or other areas as shown in the voltage-gated proton channel, Hv1 [31].

Structural element of the EF-hand like region of ANO1

EF-hand like regions in Ca²⁺ sensors and buffers usually has common structural features. In particular, canonical EF-hand regions commonly contain Asp and Glu residues in their 1, 3, 5, 9, and 12 positions for Ca²⁺ coordination [22, 23]. Furthermore, EF-hand proteins have a helix-loop-helix structure, and the helix-loop-helix motifs are present in pairs. In the present study, we performed computer modeling to determine the 3-dimensional structure of the EF-hand like region of ANO1. However, this region of ANO1 does not share similarity with the 3-dimensional structure of the EF hand of calmodulin, largely because helices flanking the loop region are lacking. Furthermore, the EF-hand like region in ANO1 is not present in pairs. Thus, this region in ANO1 does not fall into the canonical EF hand category. However, the primary structure of the ANO1 EF-hand like region is closely related to that of the EF hand because acidic amino acids are aligned well with those of the canonical EF-hand like region. For example, in canonical EF hands, Asp and Glu residues at the 1, 3, 5, 9, and 12 positions provide side-chain carboxylic oxygens for Ca²⁺ coordination [22, 23]. In addition, the backbone carbonyl oxygen at position 7 is also essential for Ca²⁺ chelation. Likewise, in the EF-hand like region of ANO1, Asp and Glu residues are present at the 1, 3, 5, 10, and 13 positions, which also presumably provide side-chain carboxylic oxygens for Ca²⁺ coordination. In addition, Glu at position 7 also provides a side-chain carboxylic oxygen for Ca²⁺ coordination. Thus, the structural element in this region of ANO1 explains why this region is essential for the activation by Ca²⁺⁺ (page 20~21).

This research was supported by a grant from the National Research Foundation (NRF) of Korea (NRF-2018R1D-1A1B07049840) and the National Research Foundation of Korea, funded by the Ministry of Science and ICT (2011-0018358). Also, this work was partly supported by the GRRC program of Gyeonggi province (GRRC-CHA2017-A02, Validity and Safety Evaluation of Regional Specialized Resources).