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APPLICATION NOTE
hERG K+ channel currents and
pharmacology using the IonFlux system

Introduction

HERG (human ether-a go-go-related gene) K+ channels are strongly expressed in the
heart and are responsible for a rapid component (IKr) of the repolarizing currents in the
cardiac action potential (Curran ‘95; Sanguinetti ‘95). Loss of function mutations affect-
ing hERG are associated with some inherited forms of long QT syndrome (LQTS) and
increase the risk for a serious ventricular arrhythmia, torsade de pointes (Tanaka ‘97;
Moss ‘02). HERG K+ channel inhibition by both cardiac and noncardiac drugs has also
been identified as the most common cause of acquired, drug-induced LQTS that may
lead to sudden cardiac death (Vandenberg, Walker & Campbell ‘01). In fact, the side
effect of hERG K+ channel inhibition is one of the major reasons of drug withdrawal or
drug re-labeling in recent years, therefore in vitro evaluation of the effects of drugs on
hERG channels expressed heterologously in mammalian cells has been recommended
as part of the preclinical safety package by the International Conference on Harmoniza-
tion (ICHS7B Expert Working Group, ‘02).
The gold standard of evaluating drug effects on hERG K+ current is manual
patch-clamp recording. However, this low-throughput, high-cost approach is
Figure 1. The IonFlux system utilizes a "plate reader" limiting in safety screening of large numbers of drugs. Recently, automated format to simplify workflow and increase throughput. electrophysiology systems have been developed that can obtain high-throughput Systems are available with 16 and 64 amplifiers. recordings and achieve reasonably comparable results with manual patch clamp. The Throughput of 10,000 data points per day can be IonFlux™ system developed by Fluxion Biosciences is designed to combine the achieved.
convenience and throughput of a plate reader with the performance of the traditional
patch clamp assay. Here we present results of recordings of hERG currents expressed
in mammalian cell cultures and the pharmacological inhibition profiles of a panel of drug
compounds using the IonFlux system.
Materials and Methods

Cells
Chinese hamster ovary (CHO) cells that express hERG channels under G418 selection
were utilized (Millipore PrecisION™ hERG-CHO Recombinant Cell Line, Cat# CYL3038).
Cells were cultured in Glutamax DMEM/F12 medium (Gibco, Cat# 11320) containing
10% fetal bovine serum, 1% penn-strep, and 500 g/mL G418 in a humidified, 5% CO2
atmosphere. Cells were either grown at 37ºC and then transferred to a 30ºC incubator
at least 24 hrs before the experiments, or kept at 30ºC after passaging. The cultures
were never permitted to exceed 90% confluence. For experiments, cells were released
from culture flasks using Detachin (Genlantis, San Diego, CA, Cat# T100100) and after
washing and gentle trituration, cells were suspended at a concentration of 2 - 5 million
cells per ml in extracellular solution (ECS).
Solutions & Compounds
Extracellular solution (ECS) contained (mM): NaCl 145, KCl 4, MgCl2 1, CaCl2 2, HEPES
10, dextrose 10 brought to pH 7.4 using NaOH. The intracellular solution contained
(mM): KCl 120, HEPES 10, Na2ATP 4, EGTA 10, CaCl2 5.374, MgCl2 1.75 brought to
pH 7.2 using KOH.
hERG blocker compounds were purchased from Sigma. Compounds were first dissolved
in DMSO as high concentration stock solutions (10-50 mM), then diluted in dose series
in DMSO before diluted into the final concentrations in the ECS, hence the final concen-
trations of DMSO were equal in the same dose series (0.1- 0.3%). A negative control of
of DMSO solution (0.1 - 0.3%) was always applied before compound applications, and
was found not to induce a change in current amplitudes exceeding 10%.
hERG K+ channel currents and pharmacology
APPLICATION NOTE
using the IonFlux system
Experimental protocols

Each well of the IonFlux well plate consumable was loaded with 250 L of internal solution, compound, or cell suspension. IonFlux plates
look and handle exactly like standard well plates, but the plate bottom has been replaced by a microfluidic network that connects to the wells
in a repeating pattern across the plate. After loading the plate, all flow control steps were controlled by the instrument, including cell trapping,
seal formation, whole cell break-in, compound application, and washing.
The IonFlux 16 system includes 16 fully-featured amplifiers. All 16 recording channels are simultaneously applied with the specified
voltage command waveforms. The system utilizes 20-cell ensembles for each amplifier channel to improve data consistency and success
rates. For recording hERG currents, cell ensembles were voltage clamped at a holding potential of -80 mV, and seal resistances were con-
stantly monitored by a small step to -100 mV. HERG channels were activated at +50 mV (800 ms), and the outward tail current at
-50 mV were measured by subtracting a baseline reading at -50 mV before activation from the peak outward tail current at -50 mV after
activation. A step to -120 mV (800 ms) after the -50 mV repolarization step was also included for recovery of hERG channels from inactiva-
tion (see Figure 2). In experiments studying the current-voltage relationships (IV responses), either the activation step was clamped to volt-
ages between -50 mV and 60 mV in twelve 10 mV increments, or the first repolarization step was clamped to voltages between -120 mV
and +50 mV in eighteen 10 mV increments (see Figure 3). The voltage protocol was applied every 6 s. Leak current was compensated
online using two pairs of small pulses (-80 mV to -100 mV 50 ms/50 ms). Ionic currents were sampled at 5 kHz, and recorded at room tem-
perature (20 - 23 °C).
For pharmacology studies, after hERG currents were stabilized in the data acquisition phase ( 5 min), low to high concentrations of the
same compound (including negative controls of 0.1 - 0.3% DMSO solutions) were applied sequentially to the same cell ensembles for 3 - 5
minutes each, either in a cumulative fashion or in an on-off (compound-wash) sequence. During experiments, currents were continuously
monitored except during the holding periods when a holding voltage of -80 mV is applied.
Example whole-cell hERG channel currents
Figure 2 shows the waveform of the voltage protocol (top
graph) and a typical screenshot of hERG currents during
runtime at room temperature (bottom graph), in which the
traces for all 16 amplifier channels (16 cell ensembles)
were overlayed in one graph. Automatic leak compensa-
tion was turned on. Resistances and current amplitudes
(ΔI) were measured at the marked cursor positions (pink
and light green for resistance measurements, green and
blue for current measurements).
Typical currents for a 30-cell ensemble ranged from 2 - 12
nA; current amplitudes are determined by the number of
cells in the whole cell configuration and the current per cell.
The seal resistance for a 30-cell ensemble measured in
parallel ranged from 3 to 40 M, which translates to a seal
resistance
of 90–1200 M per cell (Rcell= Rensemble x 30). Figure 2. The voltage waveform for the hERG current and a representative screenshot of hERG channel recordings from 16 individual cell ensembles recorded on the same IonFlux plate. hERG K+ channel currents and pharmacology
APPLICATION NOTE
using the IonFlux system
Voltage (mV)
Activation voltage (mV)
Activation Voltage (mV) Figure 3. The current-voltage relationships of recorded hERG currents at room temperature. Cells were clamped at a holding potential of -80 mV. (A) shows a representative overlay of recording sweeps at different activation voltages from a cell ensemble. IV plots of the activation current and the normalized tail current measured at -50 mV were shown in (C) and (D) . The fully-activated IV plot for hERG cells is shown in (B) (sweep overlay) and (E) (IV plot). Voltage dependence of recorded hERG currents Current-voltage relationships of recorded hERG currents at room temperature are shown in Fig. 3. Cells were clamped at a holding poten-tial of -80 mV. Fig. 3A shows a representative overlay of recording sweeps at different activation voltages from a cell ensemble. Cells were depolarized to voltages between –60 mV and 60 mV for 800 ms to activate hERG currents, then re-polarized to -50 mV for the outward tail current. IV plots of the activation current and the normalized tail current measured at -50 mV were shown in (C) and (D). The Boltzmann function fit indicates a half maxi-mum activation voltage of – 6±0.3mV (D). This agrees well with previously presented APC data (PatchXpress, Guo & Guthrie, 2005), and is shifted by approxi-mately 8 mV from the activation voltage measured at 23 °C using manual patch clamp recording (-14mV, Zhou et al., 1998). The fully-activated IV plot for hERG cells is shown in (B) (sweep over-lay) and (E) (IV plot), where cur-rents were activated by a depolar-izing step to 50 mV, and repolar-ized to different voltages. The tail current reversed at -94 mV; at more negative voltages, the cur-rent became inward. The calcu- Figure 4. Longevity of hERG recording and two types of compound application sequences. Ex- lated reversal potential is –90 mV, perimental screen capture showing two cumulative blocker concentration sequences, followed by a based on the solutions used. protocol during which the blocker is removed before each subsequent concentration is applied. hERG K+ channel currents and pharmacology
APPLICATION NOTE
using the IonFlux system
hERG blocker IC50s Upon obtaining whole-cell access, hERG currents could be continuously recorded for more than 60 (up to 120) min (see an example in Figure 4). Therefore a couple of different methods could be used to determine IC50 value of a compound using the same cell ensemble. Each experimental pattern contains up to eight compounds that can be applied to the same cell ensemble. The example traces in Figure 4 illustrate two different dose-response experiment protocols. C1, C2, and C3 were three different concentrations from low to high (can be same or different concentrations in different zones). First, C1, C2, C3 were repeated twice in a cumula-tive fashion then in an on-off sequence (Fig. 4). Subsequent applications of the compound doses (cumulative or on-off if the block is reversible) induced similar percentage of the hERG current block at each concentration, highlighting the consistency of the IC50 studies using Ionflux-16. In this example, two different compounds (amitriptyline and cisapride) were loaded in different zones of the plate. IC50 values were generated fitting individual cell ensembles and then averaged. Figure 5. Measured IC50 values for three The IC50s of a variety of known hERG blockes with a range of lipophillicity were determined by blockers were obtained by fitting dose-response Hill fits to the data (Figure 5). Results were compared to literature values for each compound data with a Hill function. All fitting results are shown in Table 1.
Discussion

The experiments validate the applicability of the IonFlux platform for studying
L iterature IC 5 0 hERG K+ currents and screening compounds for hERG channel inhibition. Ensemble recording from multiple cells offered the advantage of greater suc-cess rates and stable currents over a long period of time during continuous recording. The activation and inactivation of the hERG current showed similar voltage-dependences as in manual patch-clamp. Due to the longevity of hERG recording (>60min), the fast application and wash-out of compounds, and the continuous monitoring of voltage-dependent currents during compound appli- cation, IC50s of the compounds can be obtained from the same set of cell ensembles in one experiment. The IC50 values obtained using the IonFlux platform agree well with literature values (Redfern ‘03; Guo & Guthrie ‘05). Even lipophilic compounds show excellent agreement with literature values: terfenadine IC50=25nM, bepredil IC50=54nM and astemizole IC50=15nM. High throughput screening of drug effects on hERG channels is critical in drug safety testing. The compound potency data obtained indicates that IonFlux will be a valuable tool for hERG screening and compound profiling. References
Curran ME, et al. (1995). Cell 80, 795-803 Dubin AE, et al. (2005) J Biomol Screen 10:168-81 Table 1. A comparison of the IC50 values for a number of Guo L & Guthrie H. (2005) J Pharmacol Toxicol Methods 52:123-35 known blockers were obtained using the IonFlux system and Moss AJ, et al. (2002) Circulation 105:794-799 compared to literature values (right column). The cLogP values Redfern WS, et al. (2003) Cardiovasc Res 58:32-45 are also shown as a measure of compound lipophilicity. Sanguinetti MC et al. (1995). Cell 81:299-307 Tanaka T, et al.(1997) Circulation 95:565-567 Vandenberg JI, Walker BD, & Campbell TJ. (2001) Trends Phamacol Sci 22:240-246 Zhou Z, et al. (1998) Biophys J 74:230-41 384 Oyster Point Blvd., #6
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Quorum sensing in aeromonas salmonicida subsp. achromogenes and the effect of the autoinducer synthase asai on bacterial virulence

Contents lists available at Veterinary Microbiology Quorum sensing in Aeromonas salmonicida subsp. achromogenes andthe effect of the autoinducer synthase AsaI on bacterial virulence Johanna Schwenteit ,, Lone Gram , Kristian F. Nielsen , Olafur H. Fridjonsson ,Uwe T. Bornscheuer Michael Givskov , Bjarnheidur K. Gudmundsdottir a Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, IS-112 Reykjavı´k, Icelandb National Food Institute, Technical University of Denmark, Søltofts Plads bldg 221, DK-2800 Kgs. Lyngby, Denmarkc DTU Sytems Biology, Technical University of Denmark, Søltofts Plads bldg 221, DK-2800 Kgs. Lyngby, Denmarkd Prokaria, Matis ohf, Vı´nlandsleið 12, 113 Reykjavı´k, Icelande Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germanyf Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark

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