NaV1.5(Late) cardiac safety assay on QPatch

Jul 27, 2020


As an alternative to standard pharmacological procedures for NaV1.5(Late)
assays, we present a more reliable and accurate NaV1.5(Late) assay on
QPatch that removes the requirement for activators like veratridine and
ATX-II and delivers improved cardiac safety screening reliability and cost.


  • High fidelity QPatch recordings of small amplitude NaV1.5
    (Late) currents from Long QT3 syndrome mutant channels.
  • Stable recordings without pharmacological activators allow
    more reliable drug potency assessment.
  • Pharmacologically validated with sodium channel blockers
    with a preference for the NaV1.5(Late)


Cardiac safety side-effects remain the major cause of new
compound attrition during the drug discovery process. This
suggests that more robust preclinical  in vitro, ex vivo and in vivo assays and models are required to predict clinical risk in humans.
Currently the industry is moving away from an over-reliance
on the human ether-a-go-go related gene (hERG) potassium
channel (also known as KV11.1) and QT prolongation readouts
by developing new initiatives that provide a more balanced
assessment of patient risk, focusing in particular on proarrhythmic
liability. The FDA’s Comprehensive in vitro Proarrhythmia Assay
(CiPA) initiative aims to more accurately model and predict
proarrhythmic risk by including data from six in vitro ion channels
(hERG, CaV1.2, NaV1.5 (Peak and Late), Kir2.1, KVLQT1 and
KV4.3) in sophisticated in silico models of human cardiac action
potentials (AP). Recently, the FDA has demonstrated that removal
of certain currents from the model affects the accuracy of
predicting proarrhythmia more than others. Mean prediction error
increased to approximately 0.25, 0.38 and 0.75 when hCaV1.2,
NaV1.5(Late) and hERG were removed, respectively (Chang et al.,

The amplitude of the NaV1.5(Late) current, also known as the
persistent current, is a small percentage (<1 %) of the peak
NaV1.5 current which undergoes bursts of channel openings
during prolonged depolarisations, such as during the adult
human cardiac AP (Chandra et al., 2018). Due to the small amplitude of NaV1.5(Late) currents, it is extremely difficult to
record in recombinant cells expressing NaV1.5. A common
solution to this problem is to use NaV1.5(Late) current enhancers,
such as veratridine or ATX-II.

However, the binding sites and
mechanism-of-action of these openers are different and their
efficacy can also vary, leading to large variations in inhibition
values of known NaV1.5(Late) current modulators. For example,
the IC50 of ranolazine is 90.8 μM in the presence of veratridine
compared with 5.4 μM when using ATX-II (Fisher et al., 2018).
Both veratridine and ATX-II are also non-selective and will
enhance currents through endogenous NaV1.x channels, which
are known to be present in CHO and HEK cells (West et al., 1992;
Lalik et al., 1993; He and Soderlund, 2010), further limiting their
pharmacological specificity for eliciting NaV1.5(Late).
Due to the small amplitude of native NaV1.5(Late) currents and
the variability, non-selectivity and costs associated with using toxin
enhancers, we created a NaV1.5(Late) cell line utilising the NaV1.5
LQT3 syndrome KPQ deletion mutant.

ATX-II increases the Nav1.5(late) currentResults and discussion
HEK vs. CHO NaV1.5 (Late) cell lines
We created polyclonal populations of ∆KPQ NaV1.5 expressing
HEK and CHO cells to compare levels of endogenous vs exogenous
current expression. The transfected CHO cells showed minimal
expression with negligible peak inward currents under standard
cell culture conditions, and < -2.0 nA of peak NaV1.5 current after
low temperature preincubation (Fig. 2). In contrast, large inward
sodium currents of ≥ -5.0 nA could be evoked from transfected
HEK cells (Fig. 2), so these were used to develop the NaV1.5(Late)
assay on QPatch.

Comparison of Nav1.5 delta KPQ peak current expression in CHO and HEK cells

Choosing the optimal voltage protocol
A number of voltage protocols have been used to evoke
NaV1.5(Late) currents, including CiPA protocols, action potential like waveform and step-ramp protocols (Fig. 3). Metrion
compared each of these voltage protocols on the HEK
NaV1.5 ∆KPQ cell line on QPatch. The CiPA step-ramp voltage
protocol produced small currents during the “ramp” phase,
but there was evidence of persistent current after the initial
depolarisation to -15 mV. Action potential-like waveforms
produced currents with a large peak current followed by a
persistent current that increased during the “repolarisation”
phase of the simulated action potential voltage command.
Finally, we tested a simple step-ramp protocol and further
optimised it to create a stable NaV1.5(Late) current assay for
pharmacological validation.

Assessment of various voltage protocols in Nav1.5 delta KPQ

Pharmacological validation of the NaV1.5 ∆KPQ assay
Metrion validated the HEK-NaV1.5(Late) cell line assay by testing
two known NaV1.5(Late) blockers using an optimised step-ramp
voltage protocol. Both mexiletine and ranolazine showed a
preference for inhibiting the late current compared with peak
inward current (Fig. 4).

Significantly, there was little difference
in the NaV1.5(Late) potency of each reference compound when
measured during the persistent phase of the long depolarising
step pulse or during the ramp command (Fig. 4).

Validation of Nav1.5 Late assay with known sodium channel inhibitors


A reliable, cost-effective and accurate NaV1.5(Late) current
assay is required on APC platforms to provide accurate cardiac
safety data to support in silico models of proarrhythmic risk.
NaV1.5(Late) assays that employ non-selective activators, such
as veratridine or ATX-II, produce unreliable IC50 values, poor
stability and can be extremely expensive (ATX-II). We have used
a pathophysiological ∆KPQ LQT3 mutant to create and validate
a NaV1.5(Late) assay that should remove the requirement for
pharmacological enhancers of NaV1.5(Late) and, thereby, deliver
improve cardiac safety screening reliability and cost.


CHO and HEK293 cells obtained from ATCC/ECACC were
transfected with a vector containing verified human LQT3 mutant
NaV1.5 ∆KPQ sequence using a liposomal based transfection
methodology. Cells were cultured and harvested using Metrion’s
optimised QPatch protocols. Standard QPatch cell suspension,
sealing and whole-cell protocols were utilized, with minor
adjustments to obtain a high proportion of gigaohm seals and
acceptable amplitude whole-cell sodium current amplitudes.


Chandra, R. et al. (2018) ‘Multiple effects of KPQ deletion mutation on gating of
human cardiac Na+ channels expressed in mammalian cells’, Am J Physiology 274,
pp. 1643–1654. PMID: 9612375

Chang, K. C. et al. (2017) ‘Uncertainty Quantification Reveals the Importance of Data
Variability and Experimental Design Considerations for in Silico Proarrhythmia Risk
Assessment’, Frontiers in Physiology, 8, p. 917. PMID: 29209226.

Fisher, J. et al. (2018) ‘Potency of Late-Nav1.5 Current Inhibtion Depends on the
Agonist Used to Augment It’, SPS 2018.

He and Soderlund (2010) ‘Human embryonic kidney (HEK293) cells express
endogenous voltage-gated sodium currents and Nav1.7 sodium channels’,
Neuroscience Letters, 469, 268-272. PMID: 20006571.

Lalik et al., (1993), ‘Characterization of endogenous sodium channel gene expressed
in Chinese hamster ovary cells’, Am J Physiology 264, pC803-809. PMID: 7682773.

Spencer, C. I. (2009) ‘Actions of ATX-II and other gating-modifiers on Na+ currents
in HEK-293 cells expressing WT and KPQ hNaV 1.5 Na channels’, Toxicon, 53, pp.
78–89. PMID: 18996139.

West et al., (1992), ‘Efficient expression of rat brain type IIA Na+ channel alpha
subunits in a somatic cell line’, Neuron 8, p59-70. PMID: 1309650.


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