Matsumura et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-020-00337-3(2021) 22:3

BMC Molecular andCell Biology

RESEARCH ARTICLE

Open AccessMechanism of hERG inhibition by gatingmodifier toxin, APETx1, deduced byfunctional characterization
Kazuki Matsumura1, Takushi Shimomura2, Yoshihiro Kubo2, Takayuki Oka3, Naohiro Kobayashi4,5, Shunsuke Imai6,
Naomi Yanase1, Madoka Akimoto1, Masahiro Fukuda1, Mariko Yokogawa1, Kazuyoshi Ikeda1, Jun-ichi Kurita7,
Yoshifumi Nishimura7, Ichio Shimada6 and Masanori Osawa1*

Abstract
Background: Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel,
the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue
peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A
previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the
VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be
conducted as only natural resources have been available until now. Therefore, it remains unclear where and how
APETx1 interacts with the VSD in the resting state.
Results: We established a method for preparing recombinant APETx1 and determined the NMR structure of therecombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild
type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1,
and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in theinhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results ofmutational analysis.
Conclusions: The present study identified the key residues of APETx1 and hERG that are involved in hERG
inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1,
which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

Background
Human ether-à-go-go-related gene potassium channel 1(hERG; KV11.1) is a voltage-gated potassium channel(KV channel) expressed in human cardiomyocytes, aswell as brain and cancer cells [1–3]. hERG conducts potassium ions (K+) across the cell membrane upon
depolarization, thereby contributing to the repolarizationof the action potential [2, 3]. This function is necessary* Correspondence: osawa-ms@pha.keio.ac.jp
1
Graduate School of Pharmaceutical Sciences, Keio University, Shibakoen,
Minato-ku, Tokyo 105-8512, Japan
Full list of author information is available at the end of the article

for a normal heartbeat, as demonstrated by the fact thatsome hERG inhibitors cause lethal arrhythmia accompanied by long QT syndrome [2, 4–6]. Recently, it has
been reported that variations in the gene encoding hERG
are associated with schizophrenia [7–9], and that alterations in hERG expression and function are observed invarious types of cancer cells and are involved in carcinogenic processes [10–12]. Non-arrhythmogenic hERG inhibitors, which block hERG without inducing arrhythmia,
can improve the survival rate among glioblastoma patients
showing high hERG expression [13]. These clinical results
demonstrate the therapeutic potential of the specific© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

ligands controlling hERG function; thus, it is of great medical importance to determine the structural mechanisms
underlying the interactions between hERG and its specificligands [3, 12, 14].
hERG is a tetrameric channel in which each subunit
comprises six transmembrane segments (S1-S6) and Nand C-terminal cytoplasmic domains [1, 15–18]. In thetetrameric architecture of KV channels, the S5 and S6
segments form a pore domain (PD) with a K+-selective
filter and an activation gate at the center of the tetramer,
and the S1-S4 segments of each subunit form a voltagesensing domain (VSD) at the four peripheries of the PD[17, 19–23]. The voltage-dependent conformationalchanges of the VSD regulate the opening and closing of
the gate in the PD [24–26]. Upon membrane
depolarization, the VSD undergoes a conformationalchange from “S4-down” to “S4-up,” in which S4 moves
from the intracellular side to the extracellular side roughly
perpendicular to the membrane plane [24–26]. To date,
three-dimensional hERG structures have been determinedby cryo-electron microscopy (cryo-EM), which has revealed that the VSD adopts the S4-up conformation under
the nominal absence of membrane potential in detergent
micelles [17]. By contrast, the resting-state structure, inwhich the VSD adopts the S4-down conformation, has
not been determined because structural analysis underresting membrane potential is technically challenging.
Specific ligands that stabilize the resting state of hERG
include APETx1, which is a 42-amino-acid peptide toxinof the sea anemone Anthopleura elegantissima [27].
APETx1 is a gating-modifier toxin that binds to the VSD
and inhibits the voltage-dependent activation of hERG,
thus stabilizing the resting-state, S4-down conformationof the VSD [27, 28]. Therefore, APETx1 is an effective
tool for characterizing the molecular surface of thehERG VSD in the S4-down conformation, and for exploring the binding sites of the specific ligands that control hERG function.
A previous study that conducted cysteine-scanning
analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1 [28]. However, only natural
resources have been available until now, and thus, mutational analysis of APETx1 could not be conducted.
Therefore, no information could be obtained regarding
the APETx1 residues crucial for hERG inhibition, and it
remains unclear how APETx1 binds to the S4-down
conformation of the VSD to inhibit hERG activation.
In the present study, we established a method for preparing recombinant APETx1 and investigated the hERG
inhibition activity of APETx1 through electrophysiological analyses of APETx1 and hERG mutants. We
identified the hydrophobic residues of APETx1, as wellas those in the hERG S3-S4 region related to hERG

Page 2 of 16

inhibition by APETx1. Next, we constructed hypothetical docking models of APETx1-VSD complex that satisfy the results of mutational analysis. These results
would help advance understanding of the inhibitory
mechanism of hERG by APETx1.

Results
Functional and structural characterization of recombinant
APETx1

In a previous study, APETx1 was purified from the sea
anemone Anthopleura elegantissima (hereafter referred
to as “natural product”) to characterize its inhibitory effect on hERG and its solution structure [27–29]. Here,
we prepared recombinant APETx1, which was expressed
as inclusion bodies in E. coli, purified in urea buffer, andrefolded by dialysis. This dialysis process allowed theformation of three intramolecular disulfide bonds (seeMaterials and Methods for details of the sample preparation). Purified recombinant APETx1 is confirmed toshow a single peak by reverse-phase high-performanceliquid chromatography (RP-HPLC; Fig. S1a). The measured molecular weight of the natural product (4551.99
Da) was consistent with the molecular weight calculatedusing the sequence with three disulfide bonds (4552.21
Da), supporting the data indicating that there are no
post-translational modifications in the natural product[27]. In the present study, the recombinant APETx1
showed a similar molecular weight (4551.267 Da; Fig.
S1b), demonstrating chemical equivalence with the natural product. Therefore, we examined whether the prepared recombinant APETx1 is also equivalent to thenatural product in terms of structure and function.
First, a set of amide proton chemical shifts of the recombinant APETx1 was compared with the corresponding set from the natural product at pH 3.0 and 280 K[29] in order to examine their structural equivalence.
We established the NMR (nuclear magnetic resonance)
assignments of the recombinant APETx1 at pH 6.0 and
298 K (Fig. S2a-c). Then, the assignments of the recombinant APETx1 at pH 3.0 and 280 K were obtained by
performing pH-titration and variable-temperature measurements (Fig. S3a-d). It should be noted that the backbone amide signals of Y5, F33, and L34 were notobserved in the 1H-15N HSQC (heteronuclear single
quantum correlation) spectrum at pH 3.0 and 280 K.
The chemical shift values of backbone amide protons
uniformly deviated by 0.12 ppm on average from thosepreviously reported (Fig. S3e) [29]. These differences areprobably due to systematic errors, such as from chemical
shift referencing. Taking into consideration these systematic differences, the chemical shift differences arewithin ±0.05 ppm, indicating that the structure of the recombinant APETx1 is identical to that of the natural
product.

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

Next, the hERG inhibition effect of the recombinant
APETx1 was evaluated and compared with that of thenatural product [27, 28]. We observed hERG K+ currents
in the presence or absence of the recombinant APETx1 bywhole-cell patch-clamp recordings and two-electrode voltage clamp (TEVC) recordings. The data obtained fromwhole-cell patch-clamp recordings (Fig. 1a-d) demonstrated that the recombinant APETx1 effectively inhibitedthe hERG currents and shifted the half-maximal activation
voltage (V1/2) values toward positive voltage in a dosedependent manner. These results are consistent withthose of previous studies [27, 28]. Similar results were obtained from TEVC recordings (Fig. S4a and c-d).
The apparent dissociation constant (Kd) values for thebinding of APETx1 to hERG were estimated based on thedata obtained from TEVC recordings, according to a previously reported method [28]. The fraction of uninhibitedcurrents (IToxin / IControl), which was derived from the tailcurrents at weak depolarizing pulses (− 30 mV) was reduced by incremental increases of APETx1, and the data
were fitted with three models, (A)-(C), as described previously (Fig. S4b) [28]. Because residual uninhibited currents
were observed even at the 10 μM concentration of
APETx1, models (A) and (C), which assume fractional
toxin-sensitive currents, fitted the data more closely thanmodel (B), which assumes a fully toxin-sensitive current;
this is consistent with the findings of a previous study[28]. It is presently unclear which model supports APETx1
binding to hERG. It should be noted that the calculated
Kd values of the three models are as follows: (A) 1.2 μM,(B) 1.7 μM, and (C) 0.23 μM; these are 12- to 14-foldhigher than Kd values from a previous study, which are:(A) 87 nM, (B) 141 nM, and (C) 16.3 nM [28]. Differencesin the Kd values between the previous and present studies
are unclear as the natural product was not available to us,
making it difficult to conduct direct comparisons between
the recombinant APETx1 and the natural product in identical experimental conditions.

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Solution structure of APETx1 at pH 6.0

We observed pH-dependent chemical shift changes inthe backbone-amide signals between pH 3.0 and pH 6.0
for the C-terminal residues V41 and D42 and the sidechain signal of R24 (Fig. S3f). These results suggest thatthe previously reported structure, determined at pH 3.0[29], might be different from that which occurs underphysiological pH conditions. We therefore determined
the three-dimensional structure of the recombinant
APETx1 at pH 6.0 (Fig. 2a and Table 1), and comparedthis with the structure at pH 3.0 (Fig. 2b) [29]. Backbone
overlay of these two structures clearly shows that thestructure of the C-terminal region is different (Fig. 2b).
At pH 6.0, the side-chain carboxyl group of D42 lies closer
to the guanidinium group of R24, possibly because the deprotonation of the former enables the formation of hydrogen bonds or electrostatic interaction with the latter. pHtitration experiments showed that the changes in chemical
shift values are reversible (data not shown). These results
indicate that although the conformation of the C-terminal
region is slightly altered according to pH conditions, theoverall structure of APETx1 remains essentially identical.
Four clustered hydrophobic residues of APETx1
contributing to hERG inhibition identified by mutational
analysis

To identify APETx1 residues that contribute to hERG inhibition, we selected 15 residues (T3, Y5, K8, F15, K18,
T19, S22, N23, R24, T27, S29, Y32, F33, L34, and D42) for
scanning mutational analysis (Fig. 3a-b), because the side
chains of these residues are exposed on the molecular surface of APETx1 and thus are assumed to contribute to thehERG inhibition by making direct interactions with hERG.
We designed alanine-substitution mutants, omitting T3. It
has been reported that the natural T3P mutant, designated
APETx3, does not inhibit hERG at a depolarization pulseof 40 mV [31], which prompted us to design a prolinesubstitution mutant. The 1H-15N HSQC spectra of all

Fig. 1 Functional and structural characterization of recombinant APETx1 in patch-clamp recordings. a, b Current traces of hERG elicited by two
different depolarization pulses, 0 mV (a) and 60 mV (b), before (black) and after (red) the administration of 10 μM APETx1. Voltage protocol is
illustrated at the bottom of each current trace. c Normalized G-V curves (mean ± SEM) of hERG in the presence or absence of different APETx1
concentrations. d The ΔV1/2 values of different concentrations of APETx1; these values indicate the toxin-induced shift of the half-maximal
activation voltage of hERG

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

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Fig. 2 Structure determination of recombinant APETx1 at pH 6.0 and 298 K. a Stereo view of the ensemble of the 20 lowest-energy structures ofrecombinant APETx1, colored as follows: heavy atoms of backbone, navy; non-cysteine side chain, magenta; cysteine side chain, orange. b Stereo
view of the overlay of the 20 structures of recombinant APETx1 at pH 6.0 and 298 K from the present study (PDB code: 7BWI; navy) and the 25
structures of the natural product at pH 3.0 and 280 K from a previous study (PDB code: 1WQK; red) [29]Table 1 Structural statistics of the APETx1 structures in the present study (recombinant protein; 20 conformers) and previous study(natural product; 25 conformers) [27]Natural product [27]

Recombinant protein

PDB code

1WQK

7BWI

Experimental conditions

pH 3.0 and 280 K

pH 6.0 and 298 K

Distance restraints
Total NOE-derived restraints

751

766

Intraresidue restraints (|i-j| = 0)

366

131

Sequential restraints (|i-j| = 1)

140

216

Short-range restraints (2 ≤ |i-j| ≤ 4)

61

94

Long-range restraints (|i-j| ≥ 5)

184

325

Disulfide bond restraints

9

12

Dihedral angle restraints

20

41

Hydrogen-bond restraints

36–

Root-mean-square deviation (RMSD) from mean coordinate structure (Å)aBackbone heavy atoms
Residues 1–42

0.82 ± 0.17

0.66 ± 0.21

Residues 2–41

0.63 ± 0.13

0.48 ± 0.12

Residues 1–42

1.28 ± 0.17

1.01 ± 0.15

Residues 2–41

1.13 ± 0.15

0.95 ± 0.14

Residues in favored regions

84.3

93.7

Residues in allowed regions

14.0

5.6

Ramachandran outliers

1.7

0.6

All heavy atoms

Analysis of the Ramachandran plot (%)baRoot-mean-square deviation (RMSD) is calculated by MOLMOL [30]. bStereochemical quality is evaluated according to
MolProbity (http://molprobity.biochem.duke.edu/)

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

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Fig. 3 hERG inhibition by APETx1 mutants. a The primary structure of APETx1, colored as follows: positively-charged residues (K and R), blue;
negatively-charged residues (D), red; hydrophobic residues (F, I, L, P, V, W, and Y), green; cysteine residues, orange; and others (G, N, S, and T),
black. Three pairs of disulfide bonds are shown with orange lines. Non-mutated residues are drawn with semi-transparent gray marker tohighlight the mutated residues. b The structure of APETx1 is shown as a ribbon representation with sticks depicting the mutated residues. c-e
Normalized G-V curves (mean ± SEM) of hERG in the presence of 10 μM APETx1 mutants expressed by different colors and symbols. Control and
WT correspond to the normalized G-V curve in the presence and absence, respectively, of 10 μM APETx1 from Fig. 1c. Based on the ΔV1/2 values,
APETx1 mutants were categorized into the groups (I)-(III). f The ΔV1/2 values of 10 μM APETx1 mutants in hERG are represented as the meanvalues ± SEM, with the number of experiments shown in parentheses. Multiple-group comparison was performed by one-way ANOVA followed
by Tukey’s test (∗, 0.01 ≤ p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001). (g) Close-up view of the hydrophobic residues that yielded group (I)mutants (boxed residues) are shown as surface representations (left) and ribbon representations with sticks depicting side chains (right). The
mutated residues are shown in bold. The distance between the Cβ atoms of F15 and Y32 is 9.4 Å, and that between F15 and F33 is 10 Å

mutants are essentially identical to that of the wild-type(WT) APETx1; the exceptions to this are the mutatedresidue and a few neighboring residues (Fig. S5). This result indicates that none of the mutations affected the overall APETx1 structure.
hERG currents were measured in the presence or absence of the APETx1 mutants by whole-cell patch clamp
recordings (Fig. 3c-f, Fig. S6). The toxin-induced V1/2
shift (ΔV1/2) value of 10 μM WT APETx1 was approximately 24 mV (ΔV1/2 = 23.9 ± 2.5 mV; Fig. 1d). The
APETx1 mutants were categorized into three groups according to the ΔV1/2 values: (I) mutants that showed significantly decreased ΔV1/2 values relative to those of WT(F15A, Y32A, F33A, and L34A; Fig. 3c and f); (II) mutants exhibiting no significant change but a decreasing
tendency in ΔV1/2 values relative to those of WT (K18A
and T19A; Fig. 3d and f); and (III) mutants showing

ΔV1/2 values nearly equal to those of WT (T3P, Y5A,
F15A, S22A, N23A, T27A, S29A, and D42A; Fig. 3e-f).
These results clearly indicate that F15, Y32, F33, andL34 play key roles in hERG inhibition. These four residues are localized on the molecular surface of APETx1,
while K18 and T19, which yielded group (II) mutants, lie
on the periphery of the group (I) site (Fig. 3g). In contrast to these two groups, the residues that yielded group(III) mutants were not located close to the four key residues but were instead dispersed on the molecular surface of APETx1 (Fig. 3b). These results suggest that themolecular surface formed by the residues F15, Y32, F33,
and L34 are foundational to the interactions between
APETx1 and hERG.
As noted above, APETx3 is reported to show no inhibitory effect on hERG [31]. Unexpectedly, our results
showed that recombinant APETx3 shifted the V1/2

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

values toward positive voltage by approximately 19 mV,
which is comparable to the effect of APETx1 (Fig. 3f).
Note, a V1/2-shift effect has not been characterized forthe native APETx3. Moreover, since the structure of native APETx3 is not reported, we are unable to confirm
whether the recombinant APETx3 is structurally identical to native APETx3. Nonetheless, the chemical shift
values of the recombinant APETx1 and APETx3 toxins
are similar (Fig. S5), suggesting that the T3 position of
APETx1 is not crucial for hERG inhibition.
Mutations of hERG residues that affect inhibitory activityof APETx1

We introduced a mutation to hERG to investigate whether
this would affect the inhibitory activity of APETx1 andthereby identify the APETx1-binding site on hERG. We
proceeded based on the generally accepted proposal thatthe extracellular side of the VSD of a voltage-gated ionchannel is targeted by gating-modifier toxins [32]. A previous study examining the APETx1-hERG interaction via
cysteine-scanning mutational analysis of G514-E519,
which are located in the S3-S4 region of the VSD, showedthat G514C and E518C mutations respectively increased
and decreased the ΔV1/2 value of 10 μM APETx1 [28].
These results suggest that these hERG residues in the S3S4 region are involved in APETx1 binding [28]. As mentioned above, APETx1 shifted the V1/2 values toward positive voltage (Fig. 1c-d and Fig. S4c-d), which supports thepossibility that APETx1 preferentially binds to hERG in its
resting state, as described previously [27, 28]. However, itremains unknown where and how APETx1 interacts with
the VSD to stabilize the resting state, S4-down conformation of hERG. Therefore, in the present study, we conducted a mutational analysis of the residues in the S3-S4
region of hERG, focusing on F508-L524, and omitting
S515-S517 and E519, which have been previously investigated (Fig. 4a-c) [28]. We also examined the mutational
effect of L433 and the charged residues (K434, E435,
E437, E438, D456, and D460) on the S1-S2 region to confirm whether the S1-S2 region contributes to hERG inhibition of APETx1 (Fig. 4a-b and d).
We measured the currents of the hERG mutants by
TEVC recordings using oocytes from Xenopus laevis. All
mutants expressed in X. laevis oocytes showed hooked
tail currents, which are characteristic features of hERG
(Figs. S7, S8, S9 and S10) [3]. This demonstrates that theoverall structures of hERG mutants are not substantiallydifferent from those of WT. It should be noted that,even in the absence of APETx1, some mutants (D456A,
D460A, D509A, E518C, and L524A) exhibited V1/2 shifts
toward positive voltage and others (L520A and L523A)
toward negative voltage (Figs. S7, S8, S9 and S10).
Therefore, the results pertaining to these mutants should
be carefully evaluated.

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We found that the ΔV1/2 values of 10 μM APETx1 in
TEVC recordings were comparable to those in patchclamp recordings in WT hERG (ΔV1/2 = 21.9 ± 1.1 mV,
in TEVC recordings; ΔV1/2 = 23.9 ± 2.5 mV, in patchclamp recordings; Figs. 1d and 4e). We confirmed thatthe ΔV1/2 values were increased by the G514C mutationand decreased by the E518C mutation (Fig. 4f), which isconsistent with the results of a previous study [28]. We
further found that the F508A and I521A mutations significantly decreased the ΔV1/2 values relative to WT,
while the I512A and G522A mutations increased thevalues (Fig. 4c and e-f). Furthermore, mutations of L433
on S1 and D460 on S2 also significantly decreased the
ΔV1/2 values relative to WT (Fig. 4d-f). These results
suggest that the S3-S4 loop plays a key role in APETx1
binding, while L433 on S1 and D460 on S2 are also involved in the hERG inhibition of APETx1.
As mentioned above, the F508, E518, and I521 mutations in the hERG S3-S4 region appear to decrease theapparent binding affinity of APETx1 (Fig. 4f). Of thesethree key mutants, it is particularly important that weinterpret the toxin-induced shifts in the activation voltage of the E518C mutant, as it demonstrated strongly
positive shifts in activation voltage even in the absenceof APETx1 (WT, V1/2 = − 13.0 ± 0.4 mV; E518C, V1/2 =5.0 ± 2.7; Fig. S9). Meanwhile, L524A, another hERG
mutation, also caused a large positive shift in activation
voltage (L524A, V1/2 = 8.3 ± 1.5; Fig. S10). However,
APETx1 shifted the activation voltage of the L524A mutant to the same extent as WT hERG. Alternatively,
L520A and L523A caused negative shifts in activation
voltage (L520A, V1/2 = − 34.4 ± 0.6 mV; L523A, V1/2 = −40.8 ± 1.2 mV; Fig. S10). However, these two mutations
did not alter the ΔV1/2 values of APETx1. Therefore, we
considered that the large perturbation of gating states inhERG activation by mutations did not significantly affectthe ΔV1/2 values of APETx1. These results suggest thatthe remarkable decrease in the APETx1 ΔV1/2 value by
the hERG E518C mutation is not only attributed to aconsequence of changing the channel gating properties,
but also reduced toxin-binding.
Construction of the docking models of APETx1-VSD
complex that satisfy the electrophysiological results

To investigate whether or not any hypothetical structure
would satisfy the results of the mutational analysis, we assumed that the key residues, inferred from the mutational
analysis, are involved in the direct interaction of the toxinchannel complex. The Cβ atoms of the four key hydrophobic residues (F15, Y32, F33, and L34) of APETx1 are
all located within 10 Å of each other (Fig. 3g), suggestingthat the interacting counterpart residues are complementarily distributed on the hERG molecular surface. In thehERG S4-up conformation, previously revealed by cryo-Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

Page 7 of 16

Fig. 4 Inhibition of hERG mutants by APETx1. a The primary structure of the hERG VSD, colored as follows: S1, yellow; S2, orange; S3, pale red;and S4, magenta. The residues that are missing in a cryo-EM structure [17] are colored with gray. Non-mutated residues are drawn with semitransparent gray marker to highlight the mutated residues. b The transmembrane domain of the cryo-EM structure of hERG with S4-up
conformation (PDB code: 5VA2) [17]. The VSDs are represented as ribbons, one of which is colored as in (a); the others are white. The PD isrepresented as a gray cartoon. c, d A detailed illustration of the hERG VSD, corresponding to the sequence shown in (a). The mutated residues
are represented as sticks. The residues in the extracellular region that are missing in the cryo-EM structure of hERG are depicted as dashed lines. eNormalized G-V curves (mean ± SEM) of hERG mutants showing ΔV1/2 values that are significantly different from those of WT, in the absence(black solid line) or presence (red solid line) of 10 μM APETx1. The fitting curves of WT in the presence (black dashed line) and absence (reddashed line) of 10 μM APETx1 are superimposed on those of the mutants. f The ΔV1/2 values of 10 μM APETx1 in hERG-mutants are represented
as the mean values ± SEM; the number of experiments is shown in parentheses. Multiple-group comparison was performed by one-way ANOVA
followed by Tukey’s test (∗, 0.01 ≤ p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001). g Close-up view of the residues that yielded mutations that
decreased the hERG inhibition by APETx1. The distance between the Cβ atoms of F508 and I521 are 11.7 Å, and the distance between F508 andE518 is at least 15 Å

EM analysis [17], the distance between the Cβ atoms of
F508 and I521 is approximately 12 Å, while that between
F508 and E518 appears to be greater than 15 Å (Fig. 4g);
such distances are not complementary to the distributionof the key APETx1 residues. However, a downward S4movement of hERG VSD may cause E518 and I521 on S4
to move closer to F508 on S3. This idea inspired us to investigate complementarity between APETx1 and hERG
VSD in the S4-down conformation.
Visualization of hERG VSD in the S4-down conformation possesses two potential limitations. First, the coordinates of the hERG S3-S4 loop (L511–E519), which

may be included in the APETx1-binding site, are missingin the cryo-EM structure and are unavailable [17]. Second, the structures of KV channels with S4-down conformation have not been previously reported. To
circumvent these challenges, we focused on the cryo-EM
structure of the closely related KV channel, rat ether-àgo-go potassium channel 1 (rEAG1), which includes thefull coordinates of S3-S4 loop [21]. In the rEAG1 structure, the VSDs are uncoupled with the pore in the presence of Ca2+-calmodulin, and the structures of the porein hERG and rEAG1 differ in open or closed gate [21].
However, the VSDs in both structures form similar S4-

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

up conformations (Fig. S11a). Therefore, we used thestructural model of rEAG1 as a template [21] for theconstruction of an S4-up model (“up” model, Fig. S11b).
We further constructed two hypothetical S4-down
models, the “one-helical-turn down” model and the“two-helical-turn down” model (Fig. S11b), in which theS4 helix is shifted toward the intracellular side by one or
two helical turns, respectively. These S4-down models
are based on the premise that in response to membrane
repolarization, the S4 helix moves toward the intracellular side, with the associated basic residues maintaining
salt bridges with the acidic residues in the S1-S3 helices[24–26, 33, 34]. Functional analysis suggests that thescheme of the gating-charge transfer in hERG activation
is similar to that of most-studied Shaker-type KV channels, in which the S4 helix moves to a roughly perpendicular position [35]. Thus, we used these simplified S4down models to verify where the key hERG residues,
F508, E518, and I521, become positioned following
downward movement of the S4 helix.
In these models, the side chains of hERG E518 andI521 are exposed and constitute the largest surface areas

Page 8 of 16

in the S4-down models (Fig. S11e and g), which is incontrast to the S4-up model (Fig. S11c). The S4-down
models show that the key hERG residues (F508, E518
and I521) are localized and form a crevice on the molecular surfaces, which appears complementary to the
distribution of the APETx1 active residues (Fig. S11f andh, left, white dotted circle). By contrast, the S4-up modelshows that the key hERG residues are not localized on
the molecular surface, nor is a crevice formed, suggesting that all key APETx1 residues cannot simultaneouslyinteract with the hERG S4-up conformation (Fig. S11d).
Next, we docked APETx1 on the two S4-down models
based on the assumptions that the key residues, identified by the mutational analyses of APETx1 and hERG,
directly interact with each other (Fig. 5). Note, theAPETx1-binding position on S3 and S4 of the hERG
VSD face the lipid interface; thus, when bound to thehERG VSD, APETx1 is reasonably accommodated on
S4, without contacting the PD or other VSDs (Fig. S12ab). We also performed a docking simulation of APETx1
on the S4-up model. However, multiple clusters of docking models with no noticeable interaction of key residues

Fig. 5 Structural models of the APETx1-VSD complex that satisfies the results of the mutational analysis. APETx1 docked to the one- and twohelical-turn-down model of hERG VSD in (a) and (b), respectively. The models exclude the S1 and S2 regions, which do not come into contact
with APETx1, for clarity. The models of the hERG S3-S4 regions are represented as molecular surfaces colored according to the residues thatyielded mutations that decreased (blue) and increased (red) inhibition by APETx1. APETx1 is represented as a ribbon with green sticks
representing the key residues involved in hERG inhibition. Close-up view of the hydrogen bond between APETx1 Y32 and hERG E518 is shown in
inset. “Open-book” representations of the interaction interfaces are also drawn as semi-transparent molecular surfaces with ribbon representation

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

were observed, making it difficult to select which model
to compare to the S4-down models (data not shown).
Figure 5 shows the mapping of the residues that are
key to hERG inhibition by APETx1 (see also Fig. S11ch). On the S4-down models, a crevice formed by F508,
E518, and I521 accommodates the F15, F33, and L34
side chains of APETx1 (Fig. S11f and h). Not only are
the distributions of these residues on each molecule
complementary in these models, but so too are theshapes of the molecular surfaces. These interactions
were primarily formed by the hydrophobic residues of
APETx1 and hERG. Outside the crevice formed in thesemodels, the side chains between APETx1 Y32 and hERG
E518 were positioned closely to allow for the potential
formation of a hydrogen bond (Fig. 5a-b, inset).
APETx1 showed stronger inhibition of the hERG
I512A mutant than WT (Fig. 4f). In the one-helical-turndown model, the sterically bulky I512 side chain lies inclose proximity to E518 (Fig. 5a, inset), suggesting that
I512 hinders access of APETx1 to E518 in the S4-down
conformation. Hence, the increased inhibition of the
I512A mutant by APETx1 could be due to the removal ofsteric hindrance, resulting in a higher binding affinity to
the S4-down conformation. Moreover, the glycine residue
mutants of hERG, G514C [28] and G522A, exhibit increased inhibition by APETx1 (Fig. 4f). These remarkable
mutational effects might be due to the structural changes
caused by the mutation of glycine residues adjacent to thecritical residues (e.g., I512 and E518), which optimizes thecontact surface for APETx1 binding.

Discussion
Structure and function of the recombinant APETx1

In the present study, we conducted structural and electrophysiological analyses of recombinant APETx1 and its
mutants. We concluded that it is structurally identical to
the natural product based on the chemical shift values ofeach under identical conditions (pH 3.0, 280 K; Fig. S3e).
The structure of the C-terminal region determined in thepresent study at pH 6.0 and 298 K was slightly different
from that of the natural product determined in a previous
study under different conditions (pH 3.0, 280 K) [29]. We
attribute this difference to variations in pH (Fig. S3f), andwe used 1H-15N HSQC spectra to confirm that this structural change is reversible.
Recombinant APETx1 effectively inhibited the hERG
currents elicited by relatively weak depolarization pulses(Fig. 1a), shifting the V1/2 values toward positive voltage
in a dose-dependent manner (Fig. 1c-d, Fig. S4c-d). This
positive V1/2-shift effect suggests that APETx1 recognizes and stabilizes the resting state of hERG, in which
the VSD adopts the S4-down conformation, as described
previously [27, 28]. It should be noted that APETx1
caused a reduction in the maximal conductance atPage 9 of 16

higher voltages and faster attenuation of the tail currents(Fig. 1b and Fig. S7), suggesting that APETx1 remains
hERG-bound even when strong depolarizing pulses areapplied. These implicative phenomena were also reported in several other gating-modifier toxins thatstabilize the S4-down conformation of voltage-gated ionchannels [32, 36–43].
A previous study reported that an increase in ΔV1/2
values occurs at a micromolar concentration of APETx1(10− 6 M), while the Kd values derived from Itoxin / Icontrol
at weak depolarizing pulses are 10− 7–10− 8 M [28]. In
our TEVC recordings, however, both a decrease in Itoxin/ Icontrol (Fig. S4b) and an increase in the ΔV1/2 values(Fig. S4d) were observed at micromolar concentrations.
The reasons for the differences between the present andprevious studies remain unclear as we were unable todirectly compare the recombinant APETx1 and the natural product in the same experimental systems.
Hydrophobic surface of APETx1 contributes to hERG
inhibition

We established a method for the preparation of recombinant APETx1, which enabled us to conduct mutational
analysis. The electrophysiological analysis using the
APETx1 mutants clearly showed that the four hydrophobic residues (F15, Y32, F33, and L34) play pivotal roles inhERG inhibition. Two hydrophilic residues, K18 and T19,
also appear to contribute to the inhibition. By mappingthese residues onto the APETx1 structure, we revealed
that they are localized on the APETx1 molecular surface(Fig. 3g), where the four hydrophobic residues are clustered at the edge of the large hydrophobic surface; this isshown in green in Fig. 3g. It is well known that manygating-modifier toxins possess a large hydrophobic surface, called a hydrophobic patch, which is reported to playa role in partitioning the cell membrane prior to the binding of the target channel [44]. A spider gating-modifier
toxin, SGTx1, uses the residues on the hydrophobic patch
to interact with both the lipid membrane and the target
channel [45, 46]. It has not been reported whether thehydrophobic patch on APETx1 contributes to suchmembrane-partitioning and allosteric inhibition throughbinding of membrane lipids. However, the drastic decreases observed in the ΔV1/2 values of the four hydrophobic residues mutations, along with their localization on
the APETx1 surface, suggest that these residues engage indirect interactions with hERG in the membrane, which
could stabilize the hERG resting state.
Putative APETx1-binding residues of hERG

In the present study, we identified four previously undescribed hERG residues in the S3-S4 region (F508, I512,
I521, and G522); mutations of these, along with the previously reported residues G514 and E518 [28], affectMatsumura et al. BMC Molecular and Cell Biology(2021) 22:3

hERG inhibition of APETx1 (Fig. 4f). These results showthat the structure of the S3-S4 region plays a key role in
APETx1-binding.
A key hERG mutation, E518C, caused the large positive
shifts in the hERG activation voltage (Fig. S9). Other mutations, L520A, L523A, and L524A, which perturb the gating state in hERG activation, have only minimal effects on
the ΔV1/2 values of APETx1 (Fig. 4f and Fig. S10). Basedon these results, we determined that E518 is likely theresidue that is targeted by APETx1, as well as F508 andI521 in the present study.
Three hERG mutants, I512A, G514C, and G522A,
were more susceptible to the inhibition by APETx1 thanWT (Fig. 4f). These mutated residues are on the S3-S4
region and located near the key residues, F508, E518,
and I521, which are involved in the inhibition by
APETx1. The I512A mutation could reduce the stericbulkiness of the side chain of isoleucine in the position
around the key residues. The mutations, G514C and
G522A, would affect the local structure of the key residues. Therefore, these mutations increased the inhibition
by APETx1, which supports the idea that APETx1 recognizes the S3-S4 region of hERG.
Limitation of the APETx1-VSD complex models

One of the best ways to obtain pairwise information on
the interacting residues between hERG and APETx1 isthrough thermodynamic mutant cycle analysis [47].
However, this analysis could not be conducted in thecontext of the hERG-APETx1 interaction because even asingle mutation in either APETx1 or hERG decreases
the ΔV1/2 values to nearly zero in the presence of 10 μM
APETx1 or its mutant (Figs. 3f and 4f); thus, the additiveeffect of the double mutation cannot be evaluated.
Our docking models of the APETx1-VSD complex in
the S4-down conformation were constructed to investigate whether any hypothetical structure would satisfy
the results of the mutational analysis (Fig. 5a-b). Thesemodels are based on the assumption that the key residues identified in the mutational analysis interact witheach other directly. Moreover, although the S4movement in hERG activation is characterized by several
functional analyses, the corresponding structural properties remain unclear [17, 35, 48]. We therefore constructed simplified S4-down models by shifting one or
two helical turns downward. Although, we concede that
it is not possible to determine which model is favorable
using only calculated parameters, such as buried surface
areas, the models constructed here seem to satisfy ourmutational results. Further experimental validation andmolecular dynamics simulations are required to verify
these models.
In the present study, recombinant APETx3 showed a
ΔV1/2 value comparable to that of APETx1 (Fig. 3f).

Page 10 of 16

Although direct comparison between the recombinant
and native APETx3 is required to reveal whether this result can be applied to the native APETx3, we have successfully demonstrated that T3 of APETx1 has little
involvement in hERG inhibition, which is consistent
with our model, in which T3 of APETx1 does not directly contact the hERG VSD.
The mutational analysis of hERG clearly shows that the
S3-S4 region is crucial for inhibition by APETx1, but wecannot rule out the possibility that an additional hERG region is also involved in binding with APETx1. In particular, the mutations of L433 on S1 and D460 on S2
decreased inhibition by APETx1 (Fig. 4f-g). Althoughthese residues do not make direct contact with APETx1 in
our models (Fig. S12a-b), their mutations might affect thestructure of the APETx1-binding site in the S3-S4 region.

Conclusions
The present study identified the key residues of APETx1and hERG that are involved in hERG inhibition by
APETx1. Based on the assumption that the distributionof these residues on each molecule is complementary,
we built structural models of APETx1-VSD complex
that satisfy the electrophysiological results. The identified key residues will help advance understanding of theinhibitory mechanism of APETx1 and other gatingmodifier toxins, which could provide a structural basis
for the creation of novel types of drugs targeting the
VSDs of KV channels in the future.
Methods
Recombinant expression, purification, and refolding of
WT and mutated APETx1

The DNA sequence encoding APETx1, along with an upstream TEV-protease-recognition sequence, were cloned
into the pET-30Xa/LIC vector (Novagen). All APETx1
mutants were generated by PCR-mediated site-directedmutagenesis and confirmed by DNA sequence analysis.
The plasmids were transformed into Escherichia coli
strain C41 (λDE3) for recombinant protein production.
The cells were grown in Luria-Bertani medium supplemented with 40 mg/mL kanamycin and maintained at
37 °C with shaking at 150 rpm to an optical density of
0.6–1.0 at 600 nm. Expression of the His6-tag-fusion
proteins was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG); the cells were grown for 6–
12 h at 37 °C and then harvested by centrifugation for
10 min at 5000×g. Uniformly 15N-labelled or uniformly
13
C- and 15N-labelled APETx1 samples were preparedfor NMR experiments by growing E. coli cells in M9
minimal medium supplemented with 15NH4Cl or
15
NH4Cl and 13C6 glucose.
The cells were harvested by centrifugation and disrupted by sonication in lysis buffer (20 mM Tris-HCl,

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

Page 11 of 16

pH 8.0, and 200 mM NaCl) supplemented with 0.5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,
0.15 μM aprotinin, 1 μM E-64, and 1 μM leupeptin. The
cell pellets were prepared by centrifugation for 30 min at
10000×g. The pellets were resuspended in lysis buffer
containing 0.1% Nonidet P-40, followed by centrifugation; this procedure was repeated three times for the removal of nucleic acid. The pellets were solubilized using
the lysis buffer containing 8.0 M urea for 1–2 h at room
temperature (25 °C–27 °C). After centrifugation for 30
min at 10000×g, the His6-tag-fusion proteins were purified from the supernatant by a HIS-Select® Nickel Affinity Gel (Sigma) column. The eluted His6-tag-fusion
proteins were reduced by 2 mM dithiothreitol (DTT),
followed by dilution to 3 μM or less so as to avert aggregation during the subsequent refolding step. The reduced proteins were refolded by dialysis against a redox
buffer (3 mM reduced glutathione, 0.3 mM oxidized
glutathione, 10% glycerol, 20 mM Tris-HCl, pH 9.0, and
200 mM NaCl) until the concentration of urea was lessthan 200 mM. This internal solution was next dialyzed
against a refolding buffer (20 mM Tris-HCl, pH 9.0, and
200 mM NaCl) until the urea concentration was lessthan 10 mM. The internal solution was concentratedusing Sep-Pak® C18 cartridges (Waters), and then lyophilized. The sample was dissolved in lysis buffer and thencentrifuged to remove the aggregated proteins, which
form intermolecular disulfide bonds. After digestion with
His-tag-fused TEV protease at 25 °C, cleaved His-tag and
His-tag-fused TEV protease were removed using a HISSelect® Nickel Affinity Gel (Sigma-Aldrich) column.
APETx1 was further purified by reverse-phase highperformance liquid chromatography (RP-HPLC) using an
ODS-AM column (YMC). A linear gradient from 20 to
50% acetonitrile at a flow rate of 5.0 mL/min for 30 min
was performed using water and acetonitrile containing
0.1% trifluoroacetic acid (TFA). The eluted APETx1 wasdetected by matrix-assisted laser-desorption ionization–
time-of-flight mass spectroscopy (MALDI-TOF MS) on a
MALDI-8020 mass spectrometer (Shimadzu). α-cyano-4hydroxycinnamic acid was used for the matrix. An external calibration was performed using a ProteoMass Peptide
and Protein MALDI-MS Calibration Kit (Sigma-Aldrich).

at 298 K. The mixing times of the 15N-edited and the 13Cedited NOESY (nuclear Overhauser effect spectroscopy)
experiments, which were used for sequential assignments,
were set to 100 ms and 120 ms, respectively. 1H-15N
HSQC spectra were also measured at different pH levels(pH 3.0, pH 4.5, and pH 6.0; the phosphate buffer was pHadjusted with HCl) and temperatures (280 K, 290 K, and
298 K) using 140 μM uniformly 15N-labelled APETx1.
The 1H-15N HSQC spectra of uniformly 15N-labelled
WT APETx1 and selected mutants (T3P, K8A, F15A,
K18A, R24A, Y32A, and F33A) were measured in thephosphate buffer at 298 K; those of WT and the remaining
mutants (Y5A, T19A, S22A, N23A, T27A, S29A, L34A,
and D42A) were measured in 10% D2O (pH 6.0) at 298 K.
Sample concentrations of WT and mutants are as follows:
WT, 149 μM (in the phosphate buffer) or 102 μM (in 10%
D2O, pH 6.0); T3P, 50 μM; Y5A, 229 μM; K8A, 30 μM;
F15A, 184 μM; K18A, 31 μM; T19A, 290 μM; S22A,
102 μM; N23A, 76 μM; R24A, 19 μM; T27A, 129 μM;
S29A, 86 μM; Y32A, 408 μM; F33A, 634 μM; L34A,
258 μM; and D42A, 174 μM.
All spectra were processed using Bruker TopSpin 3.6
software or NMRPipe [49], and the data were analyzed
with Sparky (T. D. Goddard and D. G. Kneller, Sparky 3,
University of California, San Francisco, CA). The
APETx1 backbone and side-chain NMR signals were sequentially assigned using non-uniformly sampled data
for the following experiments: HNCACB, CBCA(CO)NH, HNCO, HCCH-COSY (correlation spectroscopy), HCCH-TOCSY (total correlation spectroscopy),
15
N-edited TOCSY, 15N-edited NOESY, and 13C-edited
NOESY experiments. The amide signals of T2 and L34
were not sequentially assigned due to the absence of
these signals on three-dimensional triple-resonance
NMR spectra at pH 6.0; however, they were observed on
1
H-15N HSQC spectra under low pH conditions (pH 3.0
and pH 4.5 at 298 K). 1H chemical shift values of WT
APETx1 were obtained using sodium 4,4-dimethyl-4silapentane-1-sulfonate (DSS) as a standard; 13C and 15N
chemical shift values were also corrected indirectly. The
1
H, 13C, and 15N chemical shift assignments at pH 6.0
and 298 K have been deposited in Biological Magnetic
Resonance Bank (BMRB ID: 36345).

NMR resonance assignments of APETx1 and validation ofmutants

NMR structure calculation of APETx1

Data were collected using Bruker Avance 500 or 600 spectrometers equipped with triple-resonance probes. For
NMR resonance assignments of APETx1, we measured
1
H-15N HSQC spectra, 1H-13C HSQC spectra, and nonuniformly sampled, three-dimensional NMR spectra using
258 μM uniformly 13C- and 15N-labelled APETx1 in a buffer containing 20 mM KH2PO4 (pH 6.0), 100 mM NaCl,and 10% D2O (hereafter referred to as “phosphate buffer”)

Data were collected using Bruker Avance III HD 700
spectrometers equipped with triple-resonance cryogenic
probes at 298 K. All experiments were performed using
691 μM uniformly 13C/15N-labelled APETx1 in a buffer
solution containing 20 mM KH2PO4 (pH 6.0), 100 mM
NaCl, and 10% D2O. The mixing times for the 15N-edited and the 13C-edited NOESY experiments for structural determination were set to 200 ms. These spectra
were processed using NMRPipe [49].

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

Based on the chemical shift difference between Cβ andCγ [50], we confirmed that the peptide bond of the P40
residue is cis-conformer, which is consistent with thestructure of the natural product [29]. This cis-peptide restraint was used for structure calculation. The dihedral
angle restraints were predicted using TALOS+ and werebased on the chemical shifts of 13Cα, 13Cβ, 13CO, 15Nα,
and 1HN [51]. NOE peaks were automatically pickedusing MagRO-NMRView [52–54]. The NOE peak intensity was converted to distance constraints, and structure
calculation was performed using the torsion angle dynamics program CYANA 3.98 [55]. First, we calculated
the preliminary structures without disulfide bond restraints to confirm that three pairs of cysteine residues
can be correctly formed into disulfide bonds. Next, we
calculated the structures using disulfide bond restraints
and obtained 100 structures in the final iteration. The 20
structures with the lowest target function were refinedby restrained molecular dynamics of 30 ps with Amber
12 [56]. The automated identification and superpositionof the ordered regions of the determined structures were
performed by using FitRobot ver. 1.00.07 [57]. Atomic coordinates of APETx1 and all restraint files used for the
structure calculations have been deposited in the Protein
Data Bank (PDB code: 7BWI). NMR structure ensembles
were visualized and the root mean square deviation(RMSD) values were calculated using MOLMOL [30].
Cell preparation for patch-clamp recordings

Stable HEK 293 cell lines expressing hERG (SB-HEKhERG; SB Drug Discovery Limited) were used. The cells
were cultured in Dulbecco’s Modified Eagle’s Medium(DMEM; Thermo, Gibco) supplemented with 10% fetal
bovine serum (FBS; Thermo, Gibco) and 1% penicillin/
streptomycin (Thermo, Gibco) in a humidified 5% CO2
atmosphere at 37 °C. For patch-clamp experiments, cultured cells on a polystyrene culture dish (Sumitomo
Bakelite, Tokyo, Japan) were detached by TrypLE Express (Thermo, Gibco) at 37 °C.
Automated patch-clamp recordings for mutational
analysis of APETx1

Whole-cell automated patch-clamp recordings were obtained using a SyncroPatch 384PE (Nanion Technologies
GmbH, Germany) with single-hole medium resistance(4–5.5 MΩ) borosilicate glass planar chips. Pulse generation and data collection were performed with PatchControl 384 V1.6.6 and DataControl384 V1.8.0 software.
Currents were sampled at 1 kHz. Leak subtraction was
performed based on a small voltage step at the beginningof the voltage protocol. Seal resistance was calculatedusing built-in protocols, and cells with a seal resistanceof 0.3–3 GΩ were analyzed.

Page 12 of 16

For automated patch-clamp recordings, the intracellularsolution contained 110 mM KF, 10 mM NaCl, 10 mM
KCl, 10 mM EGTA, and 10 mM HEPES-KOH (pH 7.2);
the extracellular solution contained 140 mM NaCl, 4 mM
KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose, and 10
mM HEPES-NaOH (pH 7.4). The command voltage step
took into account the fact that the use of these solutions
results in ~ 9 mV liquid junction potential. All experiments were performed at room temperature (20 °C–
25 °C). The holding membrane potential was set at − 80
mV. For the conductance-voltage (G-V) relationship experiments, hERG currents were elicited by depolarizing
voltage steps from − 80 mV to + 60 mV in 10 mV increments for 2 s, followed by a step pulse to − 40 mV for 2 s.
In order to determine the dose-response relationship,
APETx1 was added to the extracellular solution at concentrations of 0.1–10 μM. APETx1 mutants were added
to the extracellular solution at 10 μM for screening. The
voltage-pulse protocol for determining the G-V relationship was performed after the effects of APETx1 WT or
mutants reached a steady state.
Ethical approval

All animal experiments were approved by the Animal
Care Committee of the National Institutes of Natural
Sciences (an umbrella institution of National Institutefor Physiological Sciences, Tokyo, Japan), and were performed in accordance with its guidelines.
Source of animal

Xenopus laevis were purchased from Hamamatsu Animal Supply Co. (Hamamatsu, Japan) and used for oocyte
collection.
Preparation for TEVC recordingshERG was subcloned into an pSP64 plasmid, and hERG
mutants were generated by PCR-mediated site-directedmutagenesis using an In-Fusion HD Cloning Kit(Takara, Otsu, Japan) as described previously [58]. The
DNA sequences of all mutants were confirmed by sequencing. The complementary RNAs (cRNA) were transcribed from each linearized plasmid DNA using themMessage mMachine SP6 Transcription Kit (Ambion,
Austin, TX, USA). Xenopus laevis were purchased from
Hamamatsu Animal Supply Co. (Hamamatsu, Japan)
and used for oocyte collection. Preparation and injectionof X. laevis oocytes were performed as described previously [58, 59]. The total number of frogs for collecting
oocytes in the present study was ~ 20. Frogs were anesthetized by 0.15% tricaine during surgery. After depletion of oocytes, anaesthetized frogs were killed bydouble pithing. Currents were measured 1–3 d after thecRNA injection, depending on the required current
amplitude.

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

TEVC recordings for mutational analysis of hERG

TEVC recordings were performed essentially as described previously [58, 59]. Macroscopic currents wererecorded from injected oocytes under a two-electrodevoltage clamp (TEVC) using an amplifier (OC-725C;
Warner Instruments, Hamden, CT, USA), an AD-DA
converter (Digidata version 1440A; Molecular Devices,
Sunnyvale, CA, USA), and software for control and recording of the voltage clamp (pCLAMP version 10.7;
Molecular Devices). Glass microelectrodes were drawn
from borosilicate glass capillaries (Harvard Apparatus,
Cambridge, MA, USA) and filled with 3 M potassium
acetate and 10 mM KCl, pH 7.4 adjusted with HCl). The
electrode resistance was 0.2–0.8 MΩ. Oocytes expressing
cysteine-substitution mutants (G514C and E518C) wereincubated in 10 mM DTT-containing frog Ringer’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, and 0.3
mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, and 15
mM HEPES-NaOH, pH 7.4 with 0.1% penicillinstreptomycin) at 20 °C–25 °C for 15 min, and were thoroughly washed with DTT-free Ringer’s solution. All measurements were performed at 20 °C–25 °C and conductedwithin 30 min so as to avoid the formation of nonspecificdisulfide bonds [28, 60]. The bath solution was ND96 (96mM NaCl, 2 mM KCl, 1.8 mM CaCl2,1 mM MgCl2, and 5
mM HEPES-NaOH, pH 7.4). The holding membrane potential was set at − 90 mV. For hERG G-V relationship experiments (WT and mutants except for L520A andL523A), currents were elicited by depolarizing voltage step
pulses from − 80 mV to + 60 mV in 10-mV increments for
1 s, followed by a step pulse to − 60 mV for 1 s. Due to thenegatively shifted activation voltage of L520A and L523A,
the magnitude of the depolarizing step pulses was individually adjusted for each of these mutants.
To determine the Kd value of APETx1 binding tohERG, the initial APETx1 concentration in the bath solution of 0.1 μM, and the concentration was increased
cumulatively up to 10 μM. For mutational screening ofhERG, APETx1 was added to the bath solution at a concentration of 10 μM, which is maximal concentrationthat oocytes can tolerate without leakage. The voltagepulse protocol for G-V relationships was performed after
the effect of APETx1 reached a steady state.
Analysis of G-V relationships

All curve-fittings were performed using MATLAB
R2019b (MathWorks Inc., Natick, MA). The G-V curves
were fitted with the Boltzmann function:

G = Gmax ¼ 1=1 1 þ eðV 1=2 − V Þ =k þ C
where the G / Gmax is measured conductance relative tothe maximal conductance, as determined from the peakof the outward tail current at − 40 mV in TEVC

Page 13 of 16

recordings or at − 60 mV in patch-clamp recordings; theV1/2 value is the membrane potential when the G-V relationship reaches half-maximal activation; the k value isthe slope factor; and C is a constant component.
Statistical analysis

All averaged data are presented as the mean ± SEM. Then value is the number of recordings. Multiple-groupcomparison was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test using IBM
SPSS Statistics, Version 25 (IBM Corp. Armonk, NY.).
p < 0.05 was considered statistically significant (∗,0.01 ≤p < 0.05; ∗∗, 0.001 ≤ p < 0.01; ∗∗∗, p < 0.001).
Construction of the structural model of the APETx1-VSD
complex

Homology models of the hERG VSD (residues 398–545)
showing the S4-up or S4-down conformation were built
using MODELLER 9.23 [61]. The cryo-EM structure ofrEAG1 (PDB code: 5K7L) [21] was utilized as a template.
The intact sequence alignment was used for the up
model, whereas the three- and six-residue downward
shift sequence alignments of S4 were used for the oneand two-helical-turn down models, respectively.
The structural model of the APETx1-hERG complex
was generated with the HADDOCK2.4 web server [62].
The unambiguous distance restraints were tabulated sothat the key APETx1 residues (F15, Y32, F33, and L34)
and the key hERG residues (F508, E518, and I521) would
be located within 3.0 Å or less. The hydrogen bond restraints were also specified between the APETx1 Y32
and hERG E518 side chains. For rigid-body energy
minimization, 1000 structures with the 200 lowest energy solutions were generated and used for subsequentsemi-flexible simulated annealing and water refinement.
Molecular graphics figures were depicted using CueMol(http://www.cuemol.org/).

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12860-020-00337-3.
Additional file 1: Figure S1. Reverse-phase HPLC and MALDI-TOF MS
analysis of recombinant APETx1. (a) Chromatogram (left y-axis, black solidline) and gradient protocol (right y-axis, blue dashed line). (b) MALDI-TOF
mass spectrum.
Additional file 2: Figure S2. NMR resonance assignments of APETx1
on the 1H-15N HSQC and 1H-13C HSQC spectra observed at pH 6.0 and
298 K. 1H-15N HSQC spectrum (a), 1H-13C HSQC spectra for the aromatic
region (b), and for the aliphatic region (c).
Additional file 3: Figure S3. Variable-temperature and pH-titration
NMR measurements using 1H-15N HSQC. (a) Overlay of the 1H-15N HSQC
spectra at pH 6.0 under the different temperature conditions. (b-d) Overlay of the 1H-15N HSQC spectra at 298 K, 290 K, and 280 K in (b), (c), and(d), respectively, under different pH conditions. (e) 1H chemical shift differences of the backbone amide protons between recombinant APETx1
from the present study (BMRB ID: 36345) and the natural product from aMatsumura et al. BMC Molecular and Cell Biology(2021) 22:3

previous study (BMRB ID: 6370) [29] under the same conditions (pH 3.0
and 280 K). The 1H chemical shift values found in the previous study weresubtracted from those found in the present study. The proline residues,
which lack amide protons, are labeled in gray. Asterisks (*) show that theamide signal was not observed at pH 3.0 and 280 K. (f) pH-dependentchemical shift change (Δδ) values of the 1H-15N HSQC spectral signals at
298 K (b). The Δδ values were calculated using the following equation[63]: Δδ = [(Δδ1H)2 + (Δδ15N / 6.5)2]1/2 where Δδ1H and Δδ15N are thechemical shift changes in the 1H and 15N dimensions, respectively. The
proline residues, which lack amide protons, are labeled in gray. An asterisk (*) represents G1, which was not observed in the 1H-15N HSQC
spectra.
Additional file 4: Figure S4. Dose-dependent effects of APETx1 on
hERG in TEVC recordings. (a) G-V curves (mean ± SEM) of hERG in thepresence or absence of different concentrations of APETx1. (b) Doseresponse curve using the fraction of uninhibited currents at the depolarizing pulse, − 30 mV . We fitted the data with the following three models[28]: (A) four equivalent and independent binding sites per channel withfractional toxin-sensitive current, IToxin / Icontrol = Amax [Kd / (Kd + [APETx1])]4 + (1 − Amax), Kd = 1.2 μM, Amax = 0.87; (B) Four equivalent andindependent binding sites per channel with fully toxin-sensitive current,
IToxin / Icontrol = [Kd / (Kd + [APETx1])]4, Kd = 1.7 μM; (C) One binding site perchannel with fractional toxin-sensitive current, IToxin / Icontrol = Amax [Kd /(Kd + [APETx1])] + (1 − Amax), Kd = 0.23 μM, Amax = 0.93. (c) Normalized G-V
curves (mean ± SEM). (d) The ΔV1/2 values of different concentrations ofAPETx1. Data points and error bars represent the mean values ± SEM.
Additional file 5: Figure S5. 1H-15N HSQC spectra of APETx1 mutants.
All 1H-15N HSQC spectra were measured at pH 6.0 and 298 K in thefollowing solution: T3P, K8A, F15A, K18A, R24A, Y32A, or F33A mutants,
20 mM potassium phosphate (pH 6.0), 100 mM KCl, and 10% D2O; Y5A,
T19A, S22A, N23A, T27A, S29A, L34A, or D42A mutants, 10% D2O (pH 3.0);
and WT recorded in both conditions. The spectrum of each mutant issuperimposed onto that of WT under identical solution conditions. NMR
resonance assignments were labeled according to the signals of WT.
Additional file 6: Figure S6. G-V curves of hERG in the presence orabsence of 10 μM APETx1 and mutants. Normalized G-V curves (mean ±SEM) of hERG in the presence of 15 APETx1 mutants are sorted by aminoacid residue order, divided by three into the five panels.
Additional file 7: Figure S7. The current traces and G-V curves of hERG
and its mutants in the presence or absence of 10 μM APETx1. Current
traces of the hERG mutants before and after the administration of 10 μM
APETx1 (left). Voltage protocol is illustrated at the bottom of each current
trace. Current traces and voltage protocols at arbitrary potentials are
depicted with red to clearly indicate the current reduction by the
inhibitory effect of APETx1. G-V curves (mean ± SEM) of hERG mutants inthe absence (black filled circle and solid line) or presence (red filled circle
and solid line) of 10 μM APETx1 (right). The fitting curves of the WT in
the absence (black dashed line) and presence (red dashed line) of 10 μM
APETx1 are superimposed onto those of the mutants.
Additional file 8: Figure S8. The current traces and G-V curves of hERG
mutants in the presence or absence of 10 μM APETx1. Current traces of
the hERG mutants before and after the administration of 10 μM APETx1(left). Voltage protocol is illustrated at the bottom of each current trace.
Current traces and voltage protocols at arbitrary potentials are depictedwith red to clearly indicate the current reduction by the inhibitory effectof APETx1. G-V curves (mean ± SEM) of hERG mutants in the absence(black filled circle and solid line) or presence (red filled circle and solidline) of 10 μM APETx1 (right). The fitting curves of the WT in the absence(black dashed line) and presence (red dashed line) of 10 μM APETx1 aresuperimposed onto those of the mutants.
Additional file 9: Figure S9. The current traces and G-V curves of hERG
mutants in the presence or absence of 10 μM APETx1. Current traces of
the hERG mutants before and after the administration of 10 μM APETx1(left). Voltage protocol is illustrated at the bottom of each current trace.
Current traces and voltage protocols at arbitrary potentials are depictedwith red to clearly indicate the current reduction by the inhibitory effectof APETx1. G-V curves (mean ± SEM) of hERG mutants in the absence(black filled circle and solid line) or presence (red filled circle and solid

Page 14 of 16line) of 10 μM APETx1 (right). The fitting curves of the WT in the absence(black dashed line) and presence (red dashed line) of 10 μM APETx1 aresuperimposed onto those of the mutants.
Additional file 10: Figure S10. The current traces and G-V curves ofhERG mutants in the presence or absence of 10 μM APETx1. Current
traces of the hERG mutants before and after the administration of 10 μM
APETx1 (left). Voltage protocol is illustrated at the bottom of each current
trace. Current traces and voltage protocols at arbitrary potentials are
depicted with red to clearly indicate the current reduction by the
inhibitory effect of APETx1. G-V curves (mean ± SEM) of hERG mutants inthe absence (black filled circle and solid line) or presence (red filled circle
and solid line) of 10 μM APETx1 (right). The fitting curves of the WT in
the absence (black dashed line) and presence (red dashed line) of 10 μM
APETx1 are superimposed onto those of the mutants.
Additional file 11: Figure S11. Model building of the APETx1-VSD
complex. (a) Sequence alignment and stereo view of structure comparison between the VSD of hERG (The Universal Protein Resource Knowledgebase (UniProtKB) Entry: Q12809, PDB code: 5VA2) and that of rEAG1(UniProtKB Entry: Q63472, PDB code: 5K7L). hERG is colored as in Fig. 4a,and rEAG1 appears dark gray. (b) Validation of the homology models of
the hERG S3-S4 region. Ribbon representation of the cryo-EM structure ofhERG (PDB code: 5VA2) [17], up model, one- and two-helical-turn down
models (from left to right). Basic residues at positions K1-R5 on S4, andthe gating charge transfer center residue (F463) and the acidic residues(D456, D460, and D466) on S2 are represented as sticks. To clearly show
the position of S4, the Cα positions of the S2 residues are depicted withhorizontal gray dashed (D456, D460, and D466) or solid lines (F463). (c)Ribbon and semi-transparent surface representations of the up model ofthe S3-S4 region, colored as in Fig. 5. (d) Close-up view of (c). The one(e) and two-helical-turn down models (g) of the S3-S4 region are represented as ribbons and semi-transparent surfaces colored as in Fig. 5. On
the left of (f) and (h) are close-up views of (e) and (g), respectively. White
dotted circles indicate the crevice between F508, E518, and I521. On theright, docked APETx1 is also displayed as semi-transparent ribbons withthe green sticks indicating key residues involved in hERG inhibition.
Additional file 12: Figure S12. The binding location of APETx1 in atetrameric transmembrane architecture of hERG. APETx1-VSD complex
models, in which S4 adopts one- and two-helical-turn down conformations in (a) and (b), respectively, are superimposed onto the transmembrane domain of the cryo-EM of hERG (PDB code: 5VA2) [17], viewed
from within the membrane plane (left) and from the extracellular side(right). One of the four VSDs of the cryo-EM structure is substituted by
the APETx1-VSD complex model. The PD is represented as a cartoon, theVSDs as ribbons, and APETx1 as tubes. In the APETx1-docked VSD, S1 isyellow; S2, orange; S3 and S4 in (a), sky blue; and S3 and S4 in (b), cyan.

Abbreviations
hERG: human ether-à-go-go-related gene potassium channel 1; PD: poredomain; VSD: voltage-sensing domain; cryo-EM: cryo-electron microscopy;
RP-HPLC: reverse-phase high-performance liquid chromatography;
NMR: nuclear magnetic resonance; HSQC: heteronuclear single quantum
correlation; TEVC: two-electrode voltage clamp; WT: wild type; rEAG1: rat
ether-à-go-go potassium channel 1; IPTG: isopropyl β-D-1-thiogalactopyranoside; Tris: tris(hydroxymethyl)aminomethane; DTT: dithiothreitol; TEV: tobacco
etch virus; TFA: trifluoroacetic acid; MALDI-TOF MS: matrix-assisted laserdesorption ionization–time-of-flight mass spectroscopy; NOESY: nuclear
Overhauser effect spectroscopy; COSY: correlation spectroscopy; TOCSY: total
correlation spectroscopy; DSS: 4,4-dimethyl-4-silapentane-1-sulfonate;
BMRB: biological magnetic resonance bank; PDB: protein data bank;
DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; HEPE
S: 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; SEM: standard errorof the mean; ANOVA: analysis of varianceAcknowledgements
We are grateful to SB Drug Discovery Limited for providing SB-HEK-hERG. We
also thank Ms. C. Naito of the laboratory of Y. Kubo for technical support. We
would like to thank Editage (www.editage.com) for English language editing.

Matsumura et al. BMC Molecular and Cell Biology(2021) 22:3

Authors’ contributions
K.M. and M.O. conceptualized and designed the study. K.M., T.S., Y.K., T.O., S.I.,
N.Y., M.A., M.F., J.K., Y.N., I.S., and M.O. performed the experiments. K.M., T.S.,
T.O., N.K., M.Y., and M.O. analyzed the data. K.M., K.I., and M.O. performed themodel building. K.M. and M.O. mainly wrote the paper and T.S. and Y.K.
revised the paper. All authors read and approved the manuscript.

Page 15 of 16

8.

9.
Funding
This research was supported in part by Japan Society for the Promotion of
Science KAKENHI Grant Numbers JP17H03978 and JP19H04973 (to M.O.), a
grant from The Vehicle Racing Commemorative Foundation (to M.O.), a
grant from Takeda Science Foundation (to M.Y. and M.O.), and Platform
Project for Supporting Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science Research (BINDS))
from AMED under Grant Numbers JP20am0101073 (support number 0928)(to M.O.) and JP18am0101033 (support number 0004) (to Y.N.).
Availability of data and materials
The 1H, 13C, and 15N chemical shift assignments have been deposited in
Biological Magnetic Resonance Bank (BMRB ID: 36345). The atomic
coordinates of APETx1 and all restraint files used for the structure
calculations have been deposited in the Protein Data Bank (PDB code: 7BWI).
Ethics approval and consent to participate
All animal experiments were approved by the Animal Care Committee of the
National Institutes of Natural Sciences (an umbrella institution of National
Institute for Physiological Sciences, Tokyo, Japan), and were performed inaccordance with its guidelines.

10.
11.
12.

13.

14.
15.

16.

17.
Consent for publication
Not applicable.

18.

Competing interests
The authors declare that no competing interests exist.

19.

Author details
Graduate School of Pharmaceutical Sciences, Keio University, Shibakoen,
Minato-ku, Tokyo 105-8512, Japan. 2Division of Biophysics and Neurobiology,
Department of Molecular and Cellular Physiology, National Institute forPhysiological Sciences, Myodaiji, Okazaki-shi, Aichi 444-8585, Japan. 3Nanion
Technologies Japan K.K., Tokyo Laboratory, Wakamatsu-cho, Shinjuku-ku,
Tokyo 162-0056, Japan. 4Institute for Protein Research, Osaka University,
Yamadaoka, Suita-shi, Osaka 565-0871, Japan. 5NMR Science andDevelopment Division, RSC, RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama-shi,
Kanagawa 230-0045, Japan. 6Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
7
Graduate School of Medical Life Science, Yokohama City University,
Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.

20.

1Received: 14 October 2020 Accepted: 14 December 2020

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