Pharmacological Reports

https://doi.org/10.1007/s43440-021-00304-5

ARTICLE

Thallium‑sensitive fluorescent assay reveals loperamide as a new

inhibitor of the potassium channel Kv10.1
Arlet Loza‑Huerta1 · Edgar Milo1 · Arturo Picones1 · Arturo Hernández‑Cruz1,2 · Enoch Luis1,3 

Received: 5 April 2021 / Revised: 21 June 2021 / Accepted: 23 June 2021

© Maj Institute of Pharmacology Polish Academy of Sciences 2021

Abstract
Background  Ion channels have been proposed as therapeutic targets for different types of malignancies. One of the most
studied ion channels in cancer is the voltage-gated potassium channel ether-à-go-go 1 or Kv10.1. Various studies have shown
that Kv10.1 expression induces the proliferation of several cancer cell lines and in vivo tumor models, while blocking or
silencing inhibits proliferation. Kv10.1 is a promising target for drug discovery modulators that could be used in cancer
treatment. This work aimed to screen for new Kv10.1 channel modulators using a thallium influx-based assay.
Methods  Pharmacological effects of small molecules on Kv10.1 channel activity were studied using a thallium-based fluo-
rescent assay and patch-clamp electrophysiological recordings, both performed in HEK293 stably expressing the human
Kv10.1 potassium channel.
Results  In thallium-sensitive fluorescent assays, we found that the small molecules loperamide and amitriptyline exert a
potent inhibition on the activity of the oncogenic potassium channel Kv10.1. These results were confirmed by electrophysi-
ological recordings, which showed that loperamide and amitriptyline decreased the amplitude of Kv10.1 currents in a dose-
dependent manner. Both drugs could be promising tools for further studies.
Conclusions  Thallium-sensitive fluorescent assay represents a reliable methodological tool for the primary screening of
different molecules with potential activity on Kv10.1 channels or other ­K+ channels.

Keywords  Kv10.1 potassium channel · Thallium-sensitive fluorescent assay · Electrophysiology · Oncogenic channel

Abbreviations
AMI Amitriptyline
Eag1 Human ether-à-go-go potassium channel 1
HEK-WT HEK293 wild-type cells
HEK-Kv10.1 HEK293 cells stably expressing the human
Kv10.1 potassium channel
LP Loperamide
SR SR 33805 oxalate
* Enoch Luis
enoch@ifc.unam.mx; enokluis@gmail.com
1 gated potassium channels. Topologically, each subunit
Laboratorio Nacional de Canalopatías, Instituto de Fisiología
Celular, Universidad Nacional Autónoma de México,
Circuito Exterior s/n, C.U. 04510 Mexico City, Mexico
2
Departamento de Neurociencia Cognitiva, Instituto de
Fisiología Celular, Universidad Nacional Autónoma de
México, Circuito Exterior s/n, C.U. 04510 Mexico City,
Mexico
3
Cátedras CONACYT ‑ Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México, Circuito
Exterior s/n, C.U. 04510 Mexico City, Mexico
Introduction
Potassium ion channels are the most diverse family of ion
channels in mammalian cells. These transmembrane proteins
regulate many fundamental biological processes, including
generating action potentials, heart rate, electrolytic balance,
and cell division [1–3]. Given the diverse physiological roles
of ­K+ channels, they are associated with pathological con-
ditions, and consequently, are considered ideal targets for
drug discovery.
Human ether-à-go-go potassium channels (Eag1 or
Kv10.1, encoded by the KCNH1 gene) belong to voltage-
contains six transmembrane segments (S1 to S6), with
the N- and C-terminal located in the intracellular side.
The S1–S4 segments constitute the voltage-sensor domain.
S5–S6 form the selective potassium pore, and the assem-
bly of four identical subunits forms a functional Kv10.1
channel [4–6]. Opening of the channel produces a K ­ +
efflux that hyperpolarizes the resting membrane poten-
tial of cells. In healthy individuals, Kv10.1 expression is

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mainly restricted to some central brain neurons [7–9], with
practically no expression in other regions of the human
body. However, an abnormal overexpression of Kv10.1
channels has been observed in approximately 70% of solid
tumors [10–12], making them a promising target for drug
discovery modulators that would be used in cancer treat-
ment. The molecular mechanisms involved in this atypical
expression in cancer cells are not completely clear. A vast
number of publications have associated Kv10.1 overex-
pression with the malignant properties of many cancer
cells. In 1999, Luis Pardo showed for the first time that
Kv10.1 channels could transform the typical phenotype of
mammalian cells into a malignant cancer-like phenotype,
which is characterized by a high rate of proliferation and
metabolism. Furthermore, the transplant of cells express-
ing Kv10.1 channels into immunodeficient mice induced
the development of solid tumors [13]. Pharmacological
inhibition of Kv10.1 channels using small molecules or
antibodies has shown antitumor efficacy [14–20]. How-
ever, only non-specific inhibitors of Kv10.1 have been
described. Therefore, research on the Kv10.1 has focused
on searching for new and selective modulators.
Cell-based fluorescent assays have frequently been
extensively used to search and develop new modulators
of ion channels [21]. For example, to study the functional
activity of ­K+-selective ion channels, most of the fluores-
cence-based assays use (1) the permeability of thallium
­(Tl+) through open ­K+ channels, and (2) a thallium-sen-
sitive fluorescent dye [22–24]. Here, we use the ­FLIPR®
Potassium Assay (Molecular Devices) to identify new
Kv10.1 channel modulators that have not been previously
described. We found that loperamide and amitriptyline
(drugs with known action on calcium channels) exert a
potent inhibitory effect on Kv10.1 activity.
Material and methods
Culture of cells
HEK293 wild-type cells (HEK-WT) (CRL-1573, ATCC)
were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) (12800–017, Gibco) containing 10% (v/v) heat-
inactivated fetal bovine serum (26140087, Gibco) and 1%
Penicillin/Streptomycin (15140122, Gibco). HEK293 cells
stably expressing the human Kv10.1 potassium channel
(HEK-Kv10.1) (generously provided by Dr. Walter Stüh-
mer) were cultured in DMEM containing 10% fetal bovine
serum, 1% Penicillin/Streptomycin, and as selection anti-
biotic Zeocin (30 µg/ml) (R25001, Invitrogen). All cells
were cultured at 37 °C in a 5% ­CO2 incubator.
13
A. Loza‑Huerta et al.
Thallium‑sensitive fluorescent assay
Flat-bottom 96-well plates (781611, BrandTech) were
plated with HEK-WT or HEK-Kv10.1 at a density of
30,000 cells/well in 100 µl of supplemented DMEM and
incubated for 18–24 h at 37 °C in a 5% C ­ O2 incubator.
The thallium-sensitive fluorescent assay was performed
using the FLIPR Potassium assay explorer kit (R8222,
Molecular Devices) following the manufacturer’s proto-
cols. Briefly, supplemented DMEM on the 96-well plates
was removed and replaced with 50 µl of DMEM and an
equal volume of loading buffer, and then incubated at
37 °C for 60 min; loading buffer contained: 1X Hank’s
balanced salt solution (14065-056, Gibco; pH 7.4), 20 mM
HEPES, and the component A (R7578) and C (R7579) of
the FLIPR Potassium explorer kit. Next, the molecules
were pre-incubated (at a final concentration of 50 µM)
with the cells in 96-well plates for 15 min at 37 °C in a
5% ­CO2 incubator. After pre-incubation, 96-well plates
were transferred to the microplate reader FlexStation3
(Molecular Devices) controlled with the SoftMax Pro 7
software. Thallium-sensitive dye was excited at 485 nm,
and the emitted fluorescence was recorded at 515 nm. Data
were acquired at 3-s intervals for 201 s. The experimental
protocol consisted of 21 s of basal fluorescence record-
ings, after which 50 µl of the stimulus solution (2 mM
­Tl2SO4/30 mM ­K2SO4) was added, and the fluorescence
signal was recorded for another 180 s. FlexStation3 reads
the first column of the plate before moving to the next one;
every column has a positive control well (without pre-
incubated molecules) used to follow the time-dependent
variation among columns, and a negative control well
(stimulated with 2  mM ­T l 2SO 4/0  mM ­K 2SO 4) used to
correct (subtract) and determine maximal fluorescence
signal of each well (Supplemental Fig.  1A–B). Each
well’s amplitude was normalized to the positive control
well (without pre-incubated molecules) of each column.
The assay quality was estimated by Z′ factor, and only
plates with a Z′ of > 0.4 were deemed acceptable (data not
shown) [25].
Electrophysiological experiments
For patch-clamp recordings, cells were plated in 3.8 c­ m2
TC-Treated Multiple Well Plates (CLS3513, Sigma) at
200,000 cells/well. After 18–48 h, cells were trypsinized
and re-plated on 18 mm circular glass coverslips and kept
in the incubator for 1–3 h. Coverslips were transferred to
a recording chamber (RC-26G, Warner Instruments, Ham-
den, CT, USA) and whole-cell voltage-clamp recordings
were performed at room temperature and under continuous
Thallium‑sensitive fluorescent assay reveals loperamide as a new inhibitor of the potassium…
perfusion with a standard bath solution at a flow rate of
2.4 ml/min. The standard bath solution contained (in mM):
137 NaCl, 5.4 KCl, 2 ­CaCl2, 2 TEA-Cl, 10 HEPES, and 10
d -glucose (300 mOsm/kg, pH 7.4 adjusted with NaOH).
Intracellular patch-pipettes (2–3 MΩ) filling solution con-
tained (in mM): 140 KCl, 1 M ­ gCl2, 10 EGTA, 10 HEPES
(300 mOsm/kg, pH 7.2 adjusted with KOH). Ionic cur-
rents were recorded with a setup composed of Axopatch
200B/Digidata 1550/pCamp10 (amplifier/analog–digital
converter/software, all from Molecular Devices), filtered
at 5 kHz, and digitally sampled at 10 kHz. 60% of the
series resistance was electronically compensated. Small
molecules were delivered using a perfusion system VC-6
coupled to a fast-step solution charger (SF-77B, Warner
Instruments, Hamden, CT, USA).
Drugs tested on Kv10.1 currents were evaluated from a
holding voltage (Vh) of −70 mV, and currents were gener-
ated by 250 ms duration voltage steps from −70 to +50 mV
applied every 5 s. I–V curves were constructed using a proto-
col of 250 ms voltage steps from −100 to  +50 mV in 10 mV
increments and a Vh of −70 mV; the amplitude of Kv10.1
currents was measured at the end of voltage steps (aver-
age of the last 5 ms) and plotted versus voltage. Concentra-
tion–effect curves were fitted with the Hill equation using
the Levenberg–Marquardt method implemented in Origin
2016 software (OriginLab Corporation):
cn
Inhibition = Bmax , (1)
IC50n + cn
where Bmax is the maximum block, IC50 is the concentra-
tion of half-maximal inhibition, c is the concentration of the
molecules, and n is the Hill coefficient.
To determine the voltage dependence of Kv10.1 inactiva-
tion, a double-pulse protocol was used. Beginning from a
holding voltage of −70 mV, conditioning pre-pulses (Vpre) of
1.5 s in duration to voltages ranging from −130 to +50 mV
(in 20 mV increments) were applied every 10 s. After each
prepulse, a 500 ms test pulse was applied to +30 mV.
Compounds
All the small molecules (Ek-103) and maurotoxine (STM-
340) were purchased from Alomone Labs. Only astemizole
(A2861) and DMSO (D2650) were purchased from Sigma-
Aldrich. Most stock solutions were made in 100% DMSO
or water at a concentration of 100 mM. Maurotoxin stock
solution was made at 5 µM in water. All molecules were
diluted in a standard bath solution.
Data analysis
When two groups were compared, statistical significance
was assessed by Student’s t test. For multiple comparisons,
statistical significance was assessed by one-way ANOVA
followed by Games–Howell post hoc test. Differences were
considered significant when p < 0.05. In thallium assay plots
(Fig. 1D), data points for DMSO, astemizole, loperamide,
amitriptyline, and SR 33805 are displayed as mean ± SEM
of four independent experiments performed in duplicate;
the rest of the data points represent the mean ± SEM of two
independent experiments performed in duplicate. In elec-
trophysiology, data points are displayed as mean ± SEM of
n > 3 independent experiments. All data were analyzed using
OriginPro9 (OriginLab, USA), IBM SPSP Statistics version
24 and GraphPad Prism 9 (GraphPad Software, USA).
Results
Primary screening using a ­Tl+ influx assay
To set up a functional Kv10.1 thallium-sensitive fluorescent
assay, HEK-WT and HEK-Kv10.1 were plated in 96-well
plates, loaded with the thallium-sensitive dye, and assayed
for activity with a high thallium/potassium stimulus solu-
tion (2 mM ­Tl2SO4/30 mM ­K2SO4); this 2 mM ­Tl+ and
30 mM ­K+ stimulus was selected from the titration curves
(Supplemental Fig. 1C). As shown in Fig. 1A, B, HEK-WT
cells did not respond to the T­ l+/K+ stimulus; this was taken
as proof of the absence or minimal expression of voltage-
activated potassium channels. On the other hand, HEK-
Kv10.1 cells produced robust responses when the T ­ l+/K+
stimulus was applied.
Next, we did a primary screening with 21 compounds
on HEK-Kv10.1 cells to establish the fluorescent assay’s
sensitivity (Table 1); these experiments represented two
independent experiments performed in duplicate (n = 2).
Then, we obtained the median fluorescent amplitude in the
presence of each compound and determined that those com-
pounds that showed a decrease in the thallium-fluorescent
signal greater than 30% (concerning the control) would be
re-evaluated by increasing their repetitions (n). Most of the
tested compounds (50 µM) did not change the amplitude
of the responses. Only astemizole, loperamide (LP), ami-
triptyline (AMI), and SR 33805 oxalate (SR) decreased the
median fluorescent amplitude (81%, 57%, 65%, and 39%,
respectively) (Table 1). We also tested maurotoxin (100 nM),
a toxin generally used in K­ + channels assays [26], but it did
not affect Kv10.1 activity.
As we mentioned above, based on our first screening, we
increased the repetitions for astemizole, LP, AMI, SR and
DMSO (n = 4; four independent experiments performed
in duplicate). A statistical analysis using one-way ANOVA
showed astemizole (10  µM), a well-described blocker of
Kv10.1 channels [27], produce a statistically significant 82%
reduction of the fluorescence intensity (n = 4) (F5,41 = 50.13;
13

Fig. 1  Thallium-sensitive fluorescent assay for Kv10.1 channels.
A Thallium-fluorescent recordings in HEK-WT cells (red line) and
HEK-Kv10.1 cells (black line); ­Tl+ inflow through Kv10.1 channels
was induced with a stimulus solution (2 mM ­Tl2SO4/30 mM ­K2SO4,
arrow) and measured with a fluorescence FLIPR potassium assay. B
The summary bar graph of the responses in HEK-WT cells and HEK-
Kv10.1 cells. C Thallium-fluorescent responses in HEK-Kv10.1 with
control positive response (black), DMSO (orange), LP (blue), and
p < 0.0001, Games–Howell p < 0.001) (Fig.  1D). These
results confirm the functional expression of Kv10.1 chan-
nels. We evaluated the effect of DMSO (vehicle; n = 4) at
0.1% on the fluorescence responses, which did not produce
any detectable change of the high-potassium-induced thallium
signal (Fig. 1C, D) (F5,41 = 50.13; p < 0.0001, Games–How-
ell p = 0.9). We observed that LP, AMI, and SR significantly
decreased (53%, 55%, and 33%, respectively) (F5,41 = 50.13;
p < 0.0001, Games–Howell p < 0.001 for LP and RS, and
p = 0.002 for AMI) the amplitude of the fluorescence signal
mediated by Kv10.1 channels (Fig. 1C, D; Table 2).
SR 33805 oxalate produces a modest inhibition
of Kv10.1 currents
To validate the thallium assay’s results, whole-cell patch-
clamp experiments were performed in HEK-Kv10.1 cells.
13
A. Loza‑Huerta et al.
AMI (red). D The summary bar graph of the fluorescence responses
in HEK-Kv10.1 cells in the presence of different molecules (astemi-
zole was used at 10  µM, DMSO at 0.1%, Maurotoxin at 100  nM,
and the rest of the molecules were tested at a final concentration of
50 µM). Data are normalized to the positive control well. In A and C,
the solid traces and faded colors show the mean ± SEM, respectively.
Analysis of variance (ANOVA) using the Games–Howell post hoc
test: **p ≤ 0.01, ***p ≤ 0.001
The pharmacological effects of the molecules were evalu-
ated on Kv10.1-evoked currents by repeated voltages
pulses from −70 to +50 mV. First, we tested maurotoxin
(100 nM) on Kv10.1 currents, and similar to the fluores-
cent results, this toxin did not modify the amplitude of the
Kv10.1 currents (data not shown). SR has been described
as an l -type ­C a 2+ channel antagonist [28]. Various SR
concentrations were tested (0.1–300 µM). As shown in
Fig. 2A, B, 100 µM SR produced a modest (38.1%; n = 8,
p = 0.006; paired t test) and reversible inhibition on Kv10.1
currents (data not shown). To quantify SR’s potency as a
Kv10.1 inhibitor, we constructed dose-inhibition curves
(Fig. 2C) of the Kv10.1 currents evoked at +50 mV. Hill
fits were performed to these data yielding an IC50 of
130.8 ± 25.3 µM and a Hill coefficient of 1.5 ± 0.1.
Thallium‑sensitive fluorescent assay reveals loperamide as a new inhibitor of the potassium…

Table 1  Primary screening Compound Cat # Concentration n Median (range)

(µM)

DMSO A2861, Sigma-Aldrich 0.1% 2 0.97 (0.76–1.18)

Astemizole D2650, Sigma-Aldrich 10 2 0.19 (0.15–0.27)
Loperamide L-100, alomone labs 50 2 0.37 (0.28–0.65)
Amitriptyline A-155, alomone labs 50 2 0.34 (0.17–0.53)
Flunarizine F-110, alomone labs 50 2 1.0 (0.88–1.24)
Verapamil V-100, alomone labs 50 2 0.94 (0.87–0.99)
Nifedipine N-120, alomone labs 50 2 0.86 (0.78–0.94)
Lomerizine L-125, alomone labs 50 2 0.85 (0.75–0.97)
Diltiazem D-135, alomone labs 50 2 0.95 (0.88–0.99)
Felodipine F-105, alomone labs 50 2 0.97 (0.92–1.02)
Flavoxate F-140, alomone labs 50 2 0.91 (0.75–1.02)
L-651,582 L-110, alomone labs 50 2 0.94 (0.72–1.21)
Lercanidipine L-115, alomone labs 50 2 1.0 (0.86–1.19)
Nicardipine N-125, alomone labs 50 2 1.1 (0.99–1.27)
Nimodipine N-150, alomone labs 50 2 1.1 (1.05–1.32)
Nisoldipine N-160, alomone labs 50 2 1.0 (0.96–1.10)
Nitrendipine N-155, alomone labs 50 2 1.1 (0.76–1.36)
SR 33805 oxalate S-105, alomone labs 50 2 0.63 (0.53–0.67)
Maurotoxin STM-340, alomone labs 0.1 2 0.98 (0.95–1)
Amlodipine A-110, alomone labs 50 2 1.2 (0.64–1.36)
Isradipine I-100, alomone labs 50 2 0.86 (0.77–1.11)

Normalized fluorescence peak responses in the presence of different tested compounds

All median were normalized relative to the positive control well (molecules-free)

Table 2  Normalized fluorescence peak responses in the presence of to +50  mV (every 5  s) and were measured at the end
different tested compounds of each voltage step and plotted versus time (Fig. 3A).
Compound Concentration n Normalized fluores- 100  µM LP produced almost complete inhibition of
(µM) cence (mean ± SEM) Kv10.1 currents (91.3 ± 1.8%; n = 6, p < 0.0001; paired
t test), this effect was reversible upon washing out the
DMSO 0.1% 4 0.96 ± 0.04
drug (Fig. 3A–C). Figure 4A shows the dose-dependent
Astemizole 10 4 0.18 ± 0.03***
inhibitory effect of LP on Kv10.1 currents. These data
Loperamide 50 4 0.47 ± 0.05***
were fitted with a Hill equation, which yielded an IC50
Amitriptyline 50 4 0.45 ± 0.08**
of 12.8 ± 0.4 µM and a Hill coefficient of 1.2 ± 0.3. The
SR 33805 50 4 0.67 ± 0.03***
electrophysiological results with LP were consistent with
All values were normalized relative to the positive control well. One- the fluorescence thallium assays.
way ANOVA using the Games–Howell post hoc test: **p ≤ 0.01, AMI is a tricyclic antidepressant used to treat differ-
***p ≤ 0.001
ent medical conditions. As well as LP, AMI is a non-spe-
cific modulator of ion channels, including C­ a2+ and N
­ a+
Loperamide and amitriptyline produced a potent voltage-gated channels [33, 34]. As shown in Fig. 3D–F,
inhibitory effect on Kv10.1 currents 100 µM AMI produced a strong inhibitory effect on Kv10.1
currents (92.7 ± 1.1%; n = 10, p < 0.0001; paired t-test);
LP is a non-specific blocker of various ion channels and this effect was reversible upon washing (Fig. 3D–F). Fig-
receptors, including voltage-gated ­Na+, ­Ca2+, and NMDA ure 4B shows the dose-dependent inhibitory effect of AMI
receptors [29–31]. Moreover, LP is a potent agonist of on Kv10.1 currents. These data were fitted with a Hill
µ-opiate receptors (found in the gastrointestinal tract), equation, which yielded an IC50 of 15.2 ± 0.2 µM and
and it is used to treat different diarrheic syndromes [32]. a Hill coefficient of 2.2 ± 0.5. The electrophysiological
Various concentrations of LP (1–300 µM) were tested. results with AMI were also consistent with the fluores-
Kv10.1 currents were evoked by voltage steps from −70 cence thallium assay.

13

Fig. 2  SR 33805 produces a partial inhibition of Kv10.1-evoked cur-
rents. A Experimental recordings of Kv10.1 currents (at +50 mV) in
control and in the presence of SR 33805 (100 µM). Dashed lines rep-
resent 0 pA current. B Summary histogram of the effect 100 µM SR
Fig. 3  Loperamide and amitriptyline produce strong inhibition
of Kv10.1 currents. A, D The temporal course of the effect of LP
(100  µM) and AMI (100  µM) on Kv10.1-evoked currents measured
at +50  mV, respectively. B, E Experimental recordings of Kv10.1-
evoked currents (at +50  mV) in control and in the presence of LP
13
A. Loza‑Huerta et al.
on Kv10.1 currents (n = 8). C Dose-inhibition curves of SR 33805 on
Kv10.1 evoked currents (n ≥ 4 for each point). The solid lines repre-
sent the fit to the Hill equation. **p < 0.01, paired t test
(100 µM) and AMI (100 µM), respectively. Dashed lines represent 0
pA current. C, F Current–voltage relationship of Kv10.1-evoked cur-
rents and the effect of 100 µM LP (n = 6) and 100 µM AMI (n = 10),
respectively. The chemical structure of loperamide and amitriptyline
was inserted in Figs. A and D, respectively
Thallium‑sensitive fluorescent assay reveals loperamide as a new inhibitor of the potassium…
Fig. 4  Dose-inhibition curves of loperamide and amitriptyline. A, B Dose-inhibition curves for LP (n ≥ 4 for each point) and for AMI (n ≥ 3 for
each point) on Kv10.1-evoked currents. The solid lines represent the fit to the Hill equation
Loperamide accelerates voltage‑dependent
inactivation of Kv10.1 channels
Human Kv10.1 channels display a very slow voltage-
dependent inactivation. However, this biophysical prop-
erty could be pharmacologically accelerated [35]. In our
initial electrophysiological recordings, we noticed that LP
accelerates the voltage-dependent inactivation of Kv10.1.
Therefore, we decided to examine in greater detail the
effect of LP and AMI on voltage-dependent inactivation.
In the control conditions (Fig. 5A, D), the peak current
(I2) measured during the test pulse decreased slightly with
the most depolarized Vpre (+30 and +50 mV) (Fig. 5C;
data was normalized I2/I2 max). However, after 100 µM
LP was applied, we observed an acceleration in the volt-
age-dependent inactivation of Kv10.1 currents (Fig. 5B).
In the presence of LP, inactivation of Kv10.1 currents
was observed starting from a Vpre of −50  mV (n = 5;
p < 0.0042; paired t test; Fig.  5C). On the other hand,
AMI (100 µM) did not significantly affect the voltage-
dependent inactivation of Kv10.1 currents (Fig. 5D–E).
In the presence of AMI, inactivation of Kv10.1 currents
was statistically significant with a Vpre of + 30 mV (n = 6;
p = 0.0104; paired t test; Fig. 5F). Astemizole (10 µM), a
proposed pore-blocker of the Kv10.1, produces a similar
effect to LP (Supplemental Fig. 2), which indicates a simi-
lar mechanism of action.
Discussion
Patch-clamp electrophysiology is well established as the
gold-standard method for ion channel studies. However,
patch clamp could be less effective for many debatable rea-
sons for the rapid identification or assessment of active and
selective compounds on ion channels of therapeutic interest.
In recent years, there has been significant progress in devel-
oping new high-throughput technologies and non-invasive
assays to study ion channels. Indeed, cell-based fluorescent
assays have boosted ion channel drug discovery [21, 22, 24,
36].
In this work, we used a thallium-based fluorescent assay
to identify new Kv10.1 channel modulators. We found three
molecules with variable inhibitory effects on the Kv10.1
activity. The reliability of the method was proved by: (1)
no changes in fluorescence observed in HEK-WT cells, (2)
the pharmacological inhibition of the Kv10.1 channel using
the well-known blocker astemizole [27], which significantly
decreased the amplitude of the responses, and (3) patch-
clamp experiments that confirmed the inhibitory effect of
SR 33805, LP and AMI on Kv10.1 activity.
Since its discovery as an oncogenic channel, the Kv10.1
has been proposed as a therapeutic target for drug discov-
ery with possible cancer treatment use. Kv10.1 is an ion
channel overexpressed in tumor cells but with low expres-
sion in healthy tissue, therefore, the side effects of blocking
13

Fig. 5  Effect of loperamide and amitriptyline on the voltage-depend-
ent inactivation of Kv10.1 currents. A Kv10.1 currents recorded
in control solution using a double-pulse protocol shown below. B
Kv10.1 currents recorded in the presence of 100 µM LP. C For inac-
tivated curves, maximal Kv10.1 current (I2) measured at + 30  mV,
normalized (I2/I2max) and plotted as a function of Vpre, in the con-
trol condition (open circles) and in the presence of 100 µM LP (filled
circles). In control, Kv10.1 currents decreased slightly as a function
Kv10.1 could be negligible. In mammals, Kv10.1 expression
is restricted to the brain. Various studies show that the chan-
nel is found in the olfactory bulb, cerebral cortex, striatum,
hippocampus, hypothalamus, cerebellum, thalamus, and
brainstem [7–9, 37], but its physiological role is not clear.
In neurons, voltage-gated potassium channels play crucial
roles in determining the resting membrane potential and the
shape of action potentials. However, the biophysical acti-
vation properties of Kv10.1 channels indicate that they do
not participate in setting the resting membrane potential or
the shape of single-action potentials [9, 37]. Nevertheless,
in parallel fiber synapses in the cerebellar cortex, Kv10.1
modulates action potential width during high-frequency
activity [9]. Notably, Kv10.1 knockout (KO) mice do not
show apparent cognitive or behavioral differences from their
littermates control during development and adulthood [37].
The results suggest that the pharmacological inhibition of
Kv10.1 aimed at treating cancer is feasible. This approach in
conjunction with currently available treatments (e.g., chemo-
therapy) is possible and could improve cancer therapy.
To date, there are no small molecules with enough speci-
ficity on Kv10.1 activity, with most of them inhibiting other
13
A. Loza‑Huerta et al.
of the Vpre, however, in the presence of LP (n = 5), the inactivation
process of the channels was accelerated. D Kv10.1 currents recorded
in control condition. E Kv10.1 currents recorded in the presence of
AMI (100  µM). F inactivated curves in the control condition (open
diamond) and in the presence of 100 µM AMI (filled diamond; n = 6).
Dashed lines represent 0 pA current. *, ** and ***p < 0.05, < 0.01
and < 0.001 paired t test, respectively
voltage-gated ion channels, including the cardiac K ­ + chan-
nel, hERG (Kv11.1). Although none of the molecules tested
here are specific for Kv10.1 channels, the potent and revers-
ible inhibitory effects of LP on Kv10.1 make it a promising
tool for further biophysical and molecular studies and for a
better understanding of the functional role of Kv10.1 chan-
nels on cancer biology.
In the electrophysiological experiments, tested molecules
were ranked by their IC50, with the values in the follow-
ing potency sequence: loperamide > amitriptyline > SR
33805 oxalate, with the first two compounds exhibiting
an IC50 below 20 µM. Comparison with previous reports
revealed that LP can inhibit Nav1.7 channels, heterologously
expressed in HEK cells [30]. Wu et al. also reported that LP
exhibits more potent inhibition on endogenous Nav1.8 cur-
rents expressed in DRG neurons. Nevertheless, this effect
is partially mediated by the activation of µ-opioid receptors
[30]. In our experiments, the inhibitory effect of LP was
strictly on Kv10.1 channels since endogenous opioid recep-
tors are not expressed in HEK cells [38, 39]. On the other
hand, AMI is a structurally related molecule to imipramine
(another non-specific Kv10.1 blocker) and, therefore, it is
Thallium‑sensitive fluorescent assay reveals loperamide as a new inhibitor of the potassium…
not surprising that AMI also showed a considerable effect
on the Kv10.1 activity [27].
Human and mouse Kv10.1 channels are characterized by
generating almost non-inactivating macroscopic currents [4,
5], but this biophysical property could be pharmacologi-
cally accelerated [27, 35, 40]. In our electrophysiological
recordings, LP accelerates voltage-dependent inactivation
of Kv10.1 channels, and this effect was not observed in the
presence of AMI. Such differences suggest that both com-
pounds are allosteric inhibitors of the Kv10.1 channel. The
electrophysiological behavior in the presence of LP was sim-
ilar to previous reports with astemizole [27], a well-known
open-channel blocker of Kv10.1, suggesting LP could be an
open-pore inhibitor. Nevertheless, further biophysical and
pharmacological research (e.g., pharmacological competi-
tion assays or mutagenesis experiments) are needed to test
this hypothesis. We do not rule out a possible effect of LP on
the voltage-sensor domain as some other molecules acting
on ­K+ channels have shown [40, 41].
The potassium channels’ opening and closing regulate
resting membrane potential in excitable cells, modifying
the action potential frequency and duration [42]. While in
non-excitable cells, they could participate in other processes,
including the cell cycle and proliferation [43, 44]. In can-
cer cells, Kv10.1 channel overexpression is relevant for cell
proliferation [13, 45]. In contrast, their pharmacological
blockage can induce anti-proliferative effects, suggesting
a molecular link between Kv10.1 expression and the cell
cycle mechanisms. The possible connection could be related
to changes in the voltage membrane potential induced by
the aberrant expression of ion channels in cancer cells that
impact the nanoscale organization of phospholipids impli-
cated in mitogen signaling [46]. Thus, small molecules used
as pharmacological modulators of Kv10.1 channels repre-
sent a reasonable way to induce changes in the voltage mem-
brane potential that could reduce many hallmarks of cancer.
The idea of a relationship between membrane potential and
cancer has been described thoroughly in recent reviews [47,
48].
In summary, given the close correlation with the results
obtained by patch-clamp electrophysiology, the thallium-
sensitive fluorescent assay represents an effective and reli-
able method for the primary screening of different molecules
with potential activity on Kv10.1 channels. The thallium-
sensitive fluorescent assay opens the possibility to explore
the effect of peptide toxins isolated from animal venoms or
on Kv10.1 channels. Although peptide toxins exhibit high
potency and selectivity on ion channels [49], just a few of
them have been described as showing an inhibitory effect on
Kv10.1 channels [50–52].
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 43440-0​ 21-0​ 0304-5.
Author contributions  AL–H, EM and EL carried out the experiments;
EL and AH-C designed, analyzed, and interpreted the results; EL wrote
the manuscript. All the authors read and approved the final manuscript.
Funding  This work was supported by Grants: 314839, 21887 and
315803 from Consejo Nacional de Ciencia y Tecnología (CONA-
CYT) and PAPIIT-UNAM IG200119 to AH-C, and SEP-CONACYT
CB2017-2018-A1-S-13646 to E.L. We thank Fis. Cesar Oliver Lara
Figueroa and Dr. Luisa Duran Pasten for technical assistance. The Eng-
lish revision by Dr. Bristol Denlinger is also acknowledged.
Declarations 
Conflict of interest  All authors have no conflict of interest.
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