Ion channels are a viable and under- represented target class in the pharmaceutical industry. Conventional patch
clamping of a single cell at a time, the "gold standard" for studying ion channels, is labor intensive and not
amenable to screening large numbers of compounds in early stage drug discovery. As a result, surrogate measures
of ion channel activity, such as ligand displacement, ionic flux and membrane potential are used, but these assays
cannot maintain a population of ion channels in a particular conformational state due to the lack of voltage
control. Recent advances in planar patch clamp technology commenced the era of ion channel screening using a
direct electrophysiological assay, and several instruments are commercially available, including the
IonWorks® HT and PatchXpress® 7000A systems from
Molecular Devices. Although these systems offer considerably higher throughput than conventional patch clamping,
there is still a pressing need to match the throughput of surrogate assays while simultaneously driving down the
per compound assay costs. To achieve such a goal, Molecular Devices developed the IonWorks® Quattro™
system with Population Patch Clamp™ (PPC) technology.
The IonWorks® Quattro™ system from Molecular Devices—the next generation ion channel
screening tool:
Press the Play button to start the IonWorks Quattro animation.
Turn on your speakers to hear the narration. A full-screen version is
also available on the Literature & Downloads link in the right menu.
Biological variability (i.e., cell health, cell size, and channel expression levels) is the major contributing
factor for reduced success rates in planar patch clamp systems. To compensate, the IonWorks HT system was initially
designed to increase the likelihood of obtaining at least one successful recording for every compound by pipetting
compounds in quadruplicates. Thus the probability of obtaining at least one recording for every compound using a cell
line with 70% success rate is 99.2%; but at the expense of lower throughput. A four-fold increase in throughput could
be immediately realized in the IonWorks HT system by eliminating the quadruplicates. To achieve this objective,
Molecular Devices developed Population Patch Clamp™ (PPC) technology, a revolutionary approach that records
averaged ionic currents from a population of cells expressing a recombinant voltage-gated ion channel. Cells are
plated into a 384-well PatchPlate PPC™ substrate in which each well contains multiple recording sites (Figure 1).
Figure 1. The IonWorks Quattro system uses Population Patch Clamp™ technology. Each well of the PatchPlate PPC substrate contains multiple recording sites.
Because ionic currents are measured from a population of cells, the average current measured during experiments
on the IonWorks Quattro system is highly consistent from one well to the next. The measured currents on the
IonWorks Quattro system have significantly reduced coefficient of variation values. The %CV for Kv1.3
currents is 8% versus 34% for the IonWorks Quattro and IonWorks HT systems, respectively; for Nav1.5
currents, 18% versus 44%, respectively; and for hERG currents, 28% versus 50%, respectively. Raw data traces
from PPC recordings are shown in Figure 2.
Figure 2. Representative raw data current traces from Kv1.3, Nav1.5 and hERG measured on IonWorks Quattro using the PatchPlate PPC substrate.
Success rates are so high (above 95%, Table 1) that it is not necessary to apply test compounds redundantly to four
wells, thereby enabling an immediate four-fold throughput improvement over the first-generation IonWorks HT system.
Success rates for cell lines expressing Kv1.3 , Nav1.5 and hERG are summarized in Table 1.
Table 1. Comparison of success rates (%) in the IonWorks Quattro and IonWorks HT systems.
PPC
Single Hole
Ion channel (parental line)
Mean
SD
Mean
SD
Kv1.3 (CHO)
99.3
0.5
80.1
7.7
Nav1.5 (CHL)
95.5
3.2
71.3
7.1
hERG (CHO)
97.3
2.4
61.0
12.0
Two-tailed unpaired t-test; p < 0.01.
The improved data quality brings the stability and consistency that is required for robust single-point screening
and pharmacological assays for ion channel targets. Well-to-well variability is so consistent in IonWorks Quattro
experiments that highly robust Z'-Factors can be obtained, and are similar to Z'-Factor values obtained on the
FLIPR® system (Figure 3).
Figure 3. Z'-Factor for Kv1.3 pharmacology experiment on the IonWorks Quattro system. Ten-point dose-response of 4-aminopyridine (4-AP), normalized to control (pre-compound) current size. The 384-well compound plate contained 32 replicate 4-AP titrations. The Z'-Factor was calculated from 32 wells of positive controls (6 mM 4-AP) and 32 wells of negative controls (extracellular buffer only).
The increased throughput of the IonWorks Quattro system is ideal for generating comprehensive pharmacological
determinations for dozens of compounds in a single experiment. Using 96- or 384-well compound plates, a wide variety
of dose-response experiments can be designed, including any number of titrations from 3-12 concentrations—with
or without replicates. Figure 4 illustrates the tighter distribution of data from a Kv1.3 pharmacology
experiment using the IonWorks Quattro system. Each data point is comprised of 4 replicates per concentration—note
the dramatically smaller error bars in the data generated on the IonWorks Quattro system versus the IonWorks HT system.
Figure 4. Comparison of IC50 values for 4-AP inhibition of Kv1.3 channels expressed in CHO cells, obtained on the IonWorks HT (left) and IonWorks Quattro systems (right).
Potency determinations for eight reference compounds with known hERG activity (astemizole, cisapride, pimozide,
flunarizine, quinidine, terfenadine and dofetilide) were tested on the IonWorks Quattro system using the single hole
and PPC substrates. The data variability was considerable lower when using the PatchPlate PPC substrate relative to
the single hole version (Figure 5). A comparison of the potency values obtained using single hole and PPC substrates,
along with a comparison to the values reported in the literature for conventional whole-cell patch clamp
electrophysiology are summarized in Table 2. Most values, with the exception of flunarizine, are within three-fold
of the mean of the literature values reported for conventional patch clamp. Flunarizine is a slow acting compound that
did not reach steady-state inhibition even after 10 minutes of continuous stimulation using conventional patch clamp
(data not shown), which may account for the observed right shift on IonWorks Quattro, since the stimulation protocol
used was limited to five pulses over a ~5 second period.
Figure 5. Potency determinations for eight known hERG blockers on IonWorks Quattro. Comparison of values obtained using the original PatchPlate (single hole, left panel) and PatchPlate PPC (right panel) substrates. The cell plating densities were 5 x 105 cells/mL for single hole and 2 x 106 cells/mL for PPC experiments.
Table 2. Correlation of IC50 values (in µM)
for hERG blockers on the IonWork Quattro and IonWorks HT systems, and conventional patch clamp electrophysiology.
The IonWorks Quattro system is compatible with both the original single-hole PatchPlate and the new PatchPlate PPC
consumables. A new amplifier was designed for the IonWorks Quattro system allowing the user to switch between the
two PatchPlate choices. The user simply selects the type of PatchPlate while configuring the experimental protocol
in the software prior to the start of each experiment.
Population Patch Clamp technology permits a four-fold increase in throughput over the
IonWorks HT system—already the highest throughput automated electrophysiological system on the market. The
maximum number of data points per year using the IonWorks Quattro system is 400,000.
Because of the increased throughput of the IonWorks Quattro system, the cost per compound tested is significantly
reduced by more than 50% relative to the IonWorks HT system.
Ion channels are a viable and under- represented target class in the pharmaceutical industry. Conventional patch
clamping of a single cell at a time, the "gold standard" for studying ion channels, is labor intensive and not
amenable to screening large numbers of compounds in early stage drug discovery. As a result, surrogate measures
of ion channel activity, such as ligand displacement, ionic flux and membrane potential are used, but these assays
cannot maintain a population of ion channels in a particular conformational state due to the lack of voltage
control. Recent advances in planar patch clamp technology commenced the era of ion channel screening using a
direct electrophysiological assay, and several instruments are commercially available, including the
IonWorks® HT and 
