High-throughput electrophysiological assessment of drug
effect on cardiac ion channels using Qube system.
○Yuka SHIBANO1), Kazuya TSURUDOME1), Søren FRIIS2), Yuji TSURUBUCHI1)
1) Biolin Scientific K.K. 2) Biolin Scientific A/S.
Introduction
In the process of new assay determination by CiPA initiative group, it is suggested that the cardiac ion channel data from electrophysiology technique still take an important role in terms of the source of basic data for further
complemental analysis. We have released the Qube to fulfill the market requirement which is higher throughput but keep high quality of data. Take advantage of the technologies developed on QPatch, Qube utilizes the
precise pressure control and micro fluid mechanism that enable the multi-cell whole cell voltage clamp recording with high seal resistance on each cell. Here we show the examples of the currents recorded from stable cell
lines expressing cardiac ion channels on Qube.
Methods
Cells:
hERG
All hERG experiments were done with CHO hERG DUO from B’SYS. The cells were harvested using
trypsin and kept in SFM medium throughout the remaining of the day.
KvLQT1/minK
All KvLQT1/mink experiments were done with HEK KvLQT1/mink from B’SYS. The cells were harvested
using Detachin and kept in SFM medium on the Qube.
Nav1.5
All Nav 1.5 experiments were done with HEK Nav 1.5 from SB Drug Discovery. The cells were harvested
using Detachin and kept in SFM medium on the Qube.
Kv1.5
All Kv1.5 experiments were done with CHO Kv1.5 from B’SYS. The cells were harvested using trypsin and
kept in SFM medium on the Qube.
Cav1.2
All Cav 1.2 experiments were done with CHO Cav 1.2 from ChanTest. The cells were harvested using
trypsin and kept in SFM medium. This cell line is inducible – and after the induction its growth was
arrested at 30 ℃ in order to promote channel migration to the membrane. This period has to be
optimised – in these experiments we used 24 hour incubation at 30 ℃.
hERG channel
Saline
Solutions [mM]:
3 µM Dofetilide
hERG
EC: 145 NaCl, 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. Osmolality was adjusted to 320 mOsm with glucose.
IC: 130 KF, 20 KCl, 10 HEPES, 10 EGTA. pH was adjusted to 7.2 with KOH. Osmolality was adjusted to 300 mOsm with glucose.
KvLQT1/minK
EC: 145 NaCl, 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2, pH was adjusted to 7.4 with NaOH. Osmolality was adjusted to 320 with glucose.
IC: 110 K-Gluconate, 20 KCl, 10 HEPES, 5 EGTA, 2 CaCl2, 4 Na2-ATP. pH was adjusted to 7.2 with KOH. Osmolality was adjusted to 300 with glucose.
Nav1.5
EC: 145 NaCl, 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. Osmolality was adjusted to 320 mOsm with glucose.
IC: 130 CsF, 20 KCl, 10 HEPES, 10 EGTA. pH was adjusted to 7.2 with KOH. Osmolality was adjusted to 300 mOsm with glucose.
Kv1.5
EC: 145 NaCl, 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. Osmolality was adjusted to 320 mOsm with glucose.
IC: 130 K-Aspartate, 10 HEPES, 5 EGTA, 5 MgCl2, 4 Tris-ATP. pH was adjusted to 7.2 with KOH. Osmolality ws adjusted to 300 with glucose.
Cav1.2
EC for WC conformation: 145 NaCl, 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. Osmolality was adjusted to 320 mOsm
with glucose.
EC for experiments: 145 NaCl, 10 HEPES, 4 KCl, 10 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. Osmolality was adjusted to 320 mOsm with
glucose.
IC: 130 CsF, 20 CsCl, 10 HEPES, 10 EGTA. pH was adjusted to 7.2 with KOH. Osmolality was adjusted to 300 mOsm with glucose.
CaV1.2 channel
NaV1.5 channel
DMSO
Flecainide
Tetracaine
5 Hz pulse train
Nifedipine
Fig 1. Upper: typical current traces with/without dofetilide
Bottom: tail current amplitude plots
Astemizole
Bepridil
Dofetilide
E-4031
IT plot
(P1 and P10)
Fig 7. Nifedipine 6 point dose response experiment results. Sweeps with/without 0.1 µM Nifedipine (top left),
IT plot (top right), dose response hill fit (bottom left) and plate view of sweeps (bottom right).
IV curve
(P1 and P10)
Table 6. Experiment results: Average whole cell resistances, average baseline current
amplitude, IC50 and success rate for Nifedipine experiment
Fig 4. 4 point dose response in use dependent experiment results. Sweeps (top), IT plot (middle), and IV plot
(bottom) with control (0.1% DMSO), Flecainide or Tetracaine application.
Fig2. Group Hill fit results of 4 compound (Astemizole,
Bepridil, Dofetilide and E-4031)
Table 1. Experiment results: Average whole cell resistances, average baseline tail
current amplitude, IC50 and success rate for each compound including vehicle (DMSO)
Experiment Group Results
Compound name
Avg. (R-Whole Cell) [MΩ] Avg. baseline current [nA]
Astemizole
Bepridil
Dofetilide
E4031
DMSO
39.45
35.48
36.36
43.04
44.01
4.41
1.79
4.55
2.36
2.95
IC50 [M]
Success rate (%)
10.48n
358.52n
10.19n
68.98n
>100 µM
95
96
96
86
91
Table 2. Comparison of different whole cell patch clamp methods. One dose per well vs
accumulated dose response, pulse suction vs perforated to achieve whole cell patch
clamp mode, multi hole (10 cell per well) vs single hole (1 cell per well).
Experiment methods
Avg. Resistance ends
experiment [MΩ]
Avg. baseline hERG
tail current [pA]
IC50 of Cisapride
[nM]
One dose per well,
Pulse suction PC,
Multi hole (10 cells)
100
Accumulated DR,
Pulse suction PC,
Multi hole (10 cells)
115
6573
97
Accumulated DR,
Perforated PC,
Multi hole (10cells)
123
2703
138
Accumulated DR,
Pulse suction PC,
Single hole (1 cell)
772
599
63
6014
Experiment Group Results
Table 4. Experiment parameters: Average minimum resistances in last compound period, average IC50 on 1st peak,
average IC50 on 10th peak, average cell capacitance median during the experiment and success rate for each
compound including vehicle (DMSO).The whole cell patch clamp was achieved either suction pulse or perforated
usging 10µM escin in intracellular solution.
Group Name
Avg. min resistance last compound [MΩ]
Avg. IC50 P1 [µM]
Avg IC50 P10 [µM]
DMSO; suction
Flecainide; suction
Tetracaine; suction
DMSO; 10 µM
escin
Flecainide;
10 µM escin
Tetracaine;
10 µM escin
111
150
149
NA
12000
29
NA
10.56
2.51
Avg. Cell Cap.
Median [F]
95.52 p
93.07 p
93.43 p
135
NA
NA
85.57 p
93
174
3000
16.35
80.71 p
97
170
11.72
3.31
87.31 p
92
Success rate (%)
96
100
88
Compound name
Avg. (R-WholeCell) [MΩ]
Avg. baseline current [nA]
IC50 [M]
Success rate (%)
Nifedipine
34.34
2.16
181.90 n
81
NaV1.5 and hERG
channel
Baseline (saline) 10 µM Tetracaine
101
Nav1.5 only
KV1.5 channel
Nav1.5 and
hERG
KVLQT1+minK
channel
Bepridil
Multi cellular
preparation plate
30 µM HMR1556
Nav1.5 and
hERG
hERG only
Fig 5. Typical sweeps, IT plot and IV plot (left and middle). Overview of the sweep result on 384 plate (right). Current
amplitudes were measured at peak position and steady state area.
Chromanol 293B
Fig 8. Mixture of two cell lines with multiple ratio using multi cellular preparation plate: CHO-hERG
and HEK-Nav1.5 has been tested with combined voltage protocol to show the Nav1.5 and hERG
currents. The differences in current properties and effect of Tetracaine were observed.
HMR1556
Summary
Fig6. Sweeps and IT plot at the highest dose application well.
Fig 3. Current trace (top left), Group Hill fit results for 3 compounds (top middle Bepridil, bottom
left Chromanol 293B and bottom middle HMR1556), sweeps (top right) and IT plot (bottom right)
with/without 30 µM HMR1556.
Table 3. Average baseline (saline application) current size, IC50 and
success rate for each compound group.
Experiment Group Results
Compound name Baseline current [nA]
Bepridil
10.49
Chromanol 293B
9.93
HMR1556
10.91
IC50 [µM]
72.58
78.72
0.75
Success rate (%)
90
85
90
Table 5. Experiment results: Average whole cell resistances, average baseline peak current amplitude, average steady
state current, IC50 of 4-Aminopyridine, average voltage at zero current point in IV plot and success rate.
Experiment Group Results
Compound name
Avg. (R-WholeCell) [MΩ]
Avg. peak current [nA]
4-Aminopyridine
24.50
13.5
Avg. steady state current [nA] IC50 [M] Avg. V(I=0) [mV] Success rate (%)
9.7
1192 µ
-24.22
91
In this study, we have shown the results of whole cell patch clamp recordings
from cardiac ion channels. Qube performed all the experiments with high
success rates and succeeded to generate valuable data for evaluation of drug
effects on the ion channel function.
Take advantage of fine multi hole technique, Qube utilizes the pseudo multi
ion channel expressing cell experiment as we have shown here with hERG and
Nav1.5 multi-ion channel function at the same well.