Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor

Article abstract of DOI:10.1111/bph.13521

Background and Purpose: Flecainide is a use-dependent blocker of cardiac Na⁺channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na⁺channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca²⁺-release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na⁺and/or RyR2 channels. Experimental Approach: We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca²⁺sparks in intact adult rat cardiac myocytes. Key Results: Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca²⁺spark frequency. Conclusions and Implications: Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca²⁺sparks is likely, by analogy with flecainide, to result from Na⁺channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na⁺and RyR2 channels.

Full text of DOI:10.1111/bph.13521

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a
lack of direct action on the cardiac ryanodine receptor
Running title: Flecainide derivatives and ryanodine receptors
Mark L. Bannister1, Anita Alvarez-Laviada2, N. Lowri Thomas1, Sammy A. Mason1,
Sharon Coleman1, Christo L. du Plessis3, Abbygail T. Moran3, David Neill-Hall3,
Hasnah Osman4, Mark C. Bagley3, Kenneth T. MacLeod2, Christopher H. George*1,
Alan J. Williams*1
*Correspondence
Christopher H. George georgech@cardiff.ac.uk; Alan J. Williams williamsaj9@cardiff.ac.uk
1Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff, CF14 4XN,
UK
2Myocardial Function Section, National Heart and Lung Institute, Imperial College London,
London, UK
3Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton,
East Sussex, UK
4School of Chemical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.13521
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Abstract
Background and purpose
Flecainide is a use-dependent blocker of cardiac Na+ channels. The mechanisms involved in
block were elucidated using fully charged (QX-FL) and neutral (NU-FL) derivatives of
flecainide, establishing that Na+ channel block requires the cationic form of the molecule to
enter the cytosolic vestibule of the open channel. Flecainide is also effective in the treatment
of catecholaminergic polymorphic ventricular tachycardia (CPVT), however its mechanism
of action is contentious. We investigated how flecainide derivatives influence RyR2-
mediated Ca2+-release from the sarcoplasmic reticulum and whether this correlates with their
effectiveness as blockers of Na+ channels and/or RyR2.
Experimental approach
We compared the ability of QX-FL and NU-FL to block individual recombinant human
RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the
properties of Ca2+ sparks in intact adult rat cardiac myocytes.
Key results
Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal
flux of cations through RyR2 but were significantly less effective than flecainide. None of the
compounds influenced the physiologically relevant luminal to cytosol cation flux through
RyR2. Intracellular flecainide or QX-FL reduced Ca2+ spark frequency whereas NU-FL
didn’t.
Conclusions and implications
Given its inability to block physiologically-relevant cation flux through RyR2, and its lack of
efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca2+ sparks is
likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal
important differences in the nature of flecainide interaction with sites in the cytosolic
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vestibules of Na+ and RyR2 channels.
Abbreviations
CPVT catecholaminergic polymorphic ventricular tachycardia
RyR2 cardiac ryanodine receptor
SR
sarcoplasmic reticulum
TPeA tetrapentyl ammonium
TARGETS
Voltage-gated ion channelsa
Nav1.5
Table of links
Ligand-gated ion channelsb
RyR2
LIGANDS
Flecainide
These Tables list key protein targets and ligands in this article which are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the common portal for data
from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are
permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et
al., 2015a,b).
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Introduction
Flecainide is a well-characterized blocker of sarcolemmal Na+ channels (Liu et al. 2002; Liu
et al. 2003) and K+ channels (Follmer and Colatsky 1990; Paul et al. 2002). In addition,
flecainide has been shown to be effective in the treatment of CPVT and its action has been
attributed to an ability to reduce inappropriate release of Ca2+ from the SR by directly
blocking open RyR2 channels (Watanabe et al. 2009; Hilliard et al. 2010). However, in
previous studies we have established that while flecainide can act as a partial blocker of
cytosolic to luminal flux of cations in RyR2, it is unable to influence the physiologically
relevant flux of cations from the SR lumen to the cytosol that occurs during Ca2+ release. As
a consequence we have concluded that the mechanism of action of flecainide in CPVT
depends on its ability to block Na+ channels and does not involve a direct action on RyR2
(Bannister et al. 2015). This issue is important to resolve because disruption of intracellular
calcium homeostasis is also linked to dysfunction in skeletal muscle (muscular dystrophy,
malignant hyperthermia), smooth muscle (asthma), brain (stroke, Alzheimer’s disease) and
the endocrine system (type 2 diabetes mellitus). Defective RyR-mediated Ca2+ release thus
impacts on diseases of global prevalence and importance, and RyR is therefore an important
therapeutic target (see e.g. Mackrill 2010; Santulli and Marks 2015).
Block of Na+ flux into the cell by flecainide results from entry of the ligand from the cytosol
and interaction with a site in the cytosolic vestibule of the Na+ channel (Liu et al. 2003).
Similarly, flecainide interacts with an equivalent site within the cytosolic vestibule of RyR2
and gains access to this site from the cytosol (Bannister et al. 2015; Mehra et al. 2014).
Flecainide bound in RyR2 influences cation flux from the cytosolic side of the channel to the
SR lumen; a current that may contribute to charge compensation during Ca2+ release
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(Gillespie and Fill 2008), but is rapidly displaced by cations moving in the physiologically
relevant direction (from the SR lumen to the cytosol).
Given the very different abilities of flecainide to block the equivalent, physiologically
relevant, flux of cations in the Na+ and RyR2 channels, it is important to establish the
respective mechanisms governing interaction. Important information on the molecular
characteristics underlying Na+ channel block were obtained using derivatives of flecainide
with similar structural features but differing net charge (Liu et al. 2003). As highlighted in
Figure 1, at physiological pH, 100% of QX-FL will be cationic while 90% of NU-FL will be
neutral. Investigations of Na+ channel block by flecainide, QX-FL and NU-FL established
that block results from the interaction of the cationic form of the molecule with the Na+
channel (Liu et al. 2003). Flecainide has also been shown to block hERG channels at
physiologically relevant concentrations (Paul et al. 2002) and QX-FL and NU-FL have been
used to demonstrate that, as is the case in the Na+ channel, hERG block requires entry of a
cationic blocking molecule from the cell interior (Melagri et al. 2015). In the present study
we have used QX-FL and NU-FL to establish the mechanisms underlying the interaction of
flecainide with RyR2.
Our investigations demonstrate that the structural and chemical features governing flecainide
interaction with Na+ channels and RyR2 are different. While ligand charge is the primary
determinant of blocking ability in Na+ channels, small structural changes severely reduce the
ability of flecainide to block the potential charge compensating, (cytosolic to SR lumen), flux
of cations through RyR2 during Ca2+ release from the SR. Consequently, the flecainide
derivatives give us new tools with which to test the mechanism of action of flecainide in the
regulation of Ca2+ release from the SR in CPVT. Both flecainide and QX-FL (applied inside
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the cell) are effective blockers of sarcolemmal Na+ channels. Data presented here
demonstrate that while intracellular flecainide might produce some reduction in a potential
RyR2-mediated cation counter current during Ca2+ release from the SR, QX-FL will not.
Therefore, intracellular QX-FL is a Na+ channel-specific ligand and we have compared its
action with those of flecainide and NU-FL on the properties of Ca2+ sparks in intact adult rat
cardiac myocytes.
Methods
Cell culture and transfection HEK293 cells were cultured in Dulbecco’s modified Eagle
medium supplemented with 10% (v/v) foetal bovine serum, 2 mM glutamine and 100 μg.mL-
1 penicillin/streptomycin (LifeTech). Cells were incubated at 37°C, 5% CO2 and 80-90%
humidity at a density of 2 x 106 per 100 mm plate (60 cm2) 24 h prior to transfection with
pcDNA3/eGFP-hRyR2 (6 μg per 1 x 106 cells). After overnight incubation, transfected cells
were treated with sodium butyrate (2 mM) for a further 24 h before assessment of expression
(by eGFP visualisation), harvesting by centrifugation (500g AllegraR, Beckman) and storage
at -80°C.
Purification of recombinant hRyR2 channels Frozen cell pellets (typically ~50 x 106 cells)
were lysed on ice in a hypo-osmotic buffer (20 mM Tris-HCl, 5 mM EDTA; pH 7.4)
containing protease inhibitor cocktail (Roche), by passing them twenty times through a 23G
needle. Unbroken cells and nuclei were removed by low speed centrifugation (1500 g, 4°C,
AllegraR, Beckman) and the resulting lysate was subjected to a high-speed spin (100,000 g,
90 mins, 4°C, Optima L-90K, Beckman) to collect the microsomal membranes.
Solubilization of these membranes was carried out (at a concentration of 2.5 mg.mL-1) in a
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solution containing 1 M NaCl, 0.15 mM CaCl2, 0.1 mM EGTA, 25 mM PIPES, 0.6% (w/v)
CHAPS and 0.3% (w/v) phospatidylcholine, with protease inhibitor cocktail (Sigma) at 4°C.
Insoluble material was removed by centrifugation (15,000 g, 1 h at 4°C) and the supernatant
loaded onto a 5-30% (w/v) continuous sucrose gradient. Fractions containing channel
proteins were collected after overnight (18 h) centrifugation at 4°C and stored at -80°C until
use.
Conditions for recording single hRyR2 channels Single hRyR2 channels were incorporated
into bilayers formed using a suspension of phosphatidylethanolamine (Avanti Polar Lipids) in
n-decane (35 mg.mL-1). Bilayers were formed in a solution containing 610 mM KCl, 20 mM
HEPES (pH 7.4) in both (cis (0.5 mL) and trans (1 mL)) chambers. Channel incorporation
from the cis chamber was facilitated by the introduction of an osmotic gradient (using 200 μL
3 M KCl). On stirring, hRyR2 incorporates in a fixed orientation such that the cis chamber
corresponds to the cytosolic side of the channel and the trans chamber to the luminal side
(Sitsapesan and Williams 1994; Bannister et al. 2015). After channel incorporation,
symmetrical ionic conditions were re-instated by perfusion of the cis chamber with a 610 mM
KCl, 20 mM HEPES (pH 7.4) solution. All experiments were carried out at room temperature
(20-22°C). The effects of flecainide, QX-FL and NU-FL were determined after addition of
the drug to either cis or trans chambers at concentrations indicated in the text. We optimized
the quantification of block by using conditions that maximize the open duration of the
channel i.e. high permeant ion concentration (610 mM K+) in the presence of 20 μM EMD
41000, a RyR2 agonist shown previously to act via the caffeine-binding site (McGarry and
Williams 1994).
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Analysis of single channel recordings Single channel currents were low-pass filtered at 5
kHz with an 8-pole Bessel filter then digitized at 20 kHz with a PCI-6036E AD board
(National Instruments). Acquire 5.0.1. (Bruxton) was used for viewing and acquisition of the
single channel traces. Data analysis was carried out using QuB v2.0.0.13.
(www.qub.buffalo.edu). Single channel traces of 2-3 min (containing >3000 events) were
idealized using the Segmental K-means (SKM) algorithm (Qin and Li 2004) based on Hidden
Markov Models (HMM) and a dead time of 75-120 μs was imposed. In traces where substate
block was detected (by evaluation of the amplitude histogram), idealization was carried out
using a three state (closed (C) ↔ open (O) ↔ blocked (B)) scheme. QuB can accurately
distinguish between blocked and closed levels and idealization using this scheme resulted in
the calculation of mean amplitudes, open (Po), blocked (Pb) and closed (Pc) probabilities and
mean open (To), blocked (Tb) and closed (Tc) times. In all other instances where block was
not observed a 2-state (C↔O) scheme was used for idealization, which yielded amplitude, Po,
To and Tc as previously described (Mukherjee et al. 2012). Po values are shown as mean ±
SEM and fitted using non-linear regression. Rates of association (Kon) and disassociation
(Koff) were calculated as the reciprocal of To and Tb, respectively for each drug concentration
and holding potential and fitted using linear regression (through the origin when calculating
Kon for drug concentration only). Due to the large difference in voltage-dependence of block
by flecainide, QX-FL and NU-FL, Kon and Koff are plotted in a dose-normalised manner.
Although different example traces are used in Figures 2 and 4, the mean data for flecainide in
Figures 3 and 5 are the same as those published in Bannister et al., 2015. These data serve as
controls against which the actions of QX-FL and NU-FL are compared.
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Cardiomyocyte isolation Ventricular myocytes from healthy adult male Sprague Dawley rats
were enzymatically isolated as previously described (Sato et al. 2005). Briefly, hearts were
rapidly dissected and underwent retrograde perfusion using the Langendorff apparatus.
Hearts were perfused sequentially with low calcium and an enzyme (collagenase,
Worthington) solution. Ventricles were cut into small pieces and gently minced with a
Pasteur pipette. The cell suspension was filtered and cardiomyocytes were allowed to settle
by gravity.
Electrophysiological recordings combined with calcium imaging To assess the effects of
intracellularly applied drug molecules on the spatial and temporal dynamics of Ca2+ sparks,
confocal microscopy was combined with the whole-cell voltage clamp recordings. Dual
experiments were performed on adult rat ventricular myocytes at room temperature (20-22
oC). Cells were superfused with an external physiological solution of the following
composition (in mM): 137 NaCl, 10 HEPES, 10 glucose, 6 KCl, 2 MgCl2, 1 CaCl2 (pH
adjusted to 7.4 with NaOH). Borosilicate microelectrodes were pulled to give a resistance
between 2.5 and 3.5 MΩ. Vehicle, flecainide (Sigma) or its analogues QX-FL and NU-FL
(all at 5 µM) were introduced into the cell cytosol via the patch pipette. Pipettes were filled
with an intracellular solution containing either drug or vehicle in addition to membrane-
impermeant dye Fluo-3 pentapotassium salt (Invitrogen) of the following composition (in
mM): 100 K-Aspartate, 15 KCl, 10 HEPES, 5 KH2PO4, 5 Mg-ATP, 5 EGTA, 0.75 MgCl2,
0.12 CaCl2, 0.04 Fluo-3-5K+ salt (pH adjusted to 7.2 with 2 M KOH).
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After establishing a whole-cell configuration, vehicle- or drug-containing pipette solution
was allowed to equilibrate with the cytosol for 10-15 min. To facilitate steady-state SR
calcium-loading, cells underwent a series of depolarising steps of 200 ms duration to +30 mV
applied from a holding potential of -80 mV at a frequency of 3 Hz for 30 s. Ca2+ sparks were
captured following cessation of voltage-clamp stimulation. Electrophysiological recordings
were carried out using an Axopatch-1D amplifier (Axon Instruments) and a Digidata1322A
acquisition system (Axon Instruments). Series resistance and whole cell capacitance were
electronically compensated. Live cell calcium imaging was performed using a laser scanning
confocal system (BioRad Microscience Ltd, UK) and an inverted microscope (Nikon Eclipse
TE300, Nikon UK Ltd). Ca2+ imaging data were acquired and processed using
LaserSharp2000 and ImageJ (National Institutes of Health, USA) software, respectively.
Materials
QX-FL was synthesised as described (Liu et al. 2003) while NU-FL was synthesised using
the novel procedure described in Supplemental Material. The characterisation of use-
dependent block of voltage gated Na+ channels by the synthesised QX-FL and NU-FL used in
this study is also described in Supplemental Material. The drug/molecular target
nomenclature conforms to British Journal of Pharmacology's Concise Guide to Pharmacology
(Alexander et al., 2015c).
Animal welfare ethical statement
All procedures were carried out in accordance with the United Kingdom Home Office
Animals (Scientific Procedures) Act 1986, which conforms to the Guide for the Care and
Use of Laboratory Animals published by the US National Institutes of Health (NIH
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publication No. 85-23, revised 1996). Ethical approvals were granted by the United Kingdom
Home Office. Rats were sacrificed by cervical dislocation following exposure to 5%
isoflurane until the righting reflex was lost. All procedures involving animals are reported
according to the ARRIVE guidelines (Kilkenny et al., 2010).
Design and statistical analysis
Data and statistical analysis comply with the recommendations on experimental design and
analysis in pharmacology (Curtis et al., 2015). All cardiac myocytes were isolated following
the sacrifice of healthy adult male Sprague Dawley rats and there was no requirement for
randomization and blinding. For experiments using cardiac myocytes under voltage-clamp
conditions, after an initial assessment of Ca2+ handling in control (drug-naïve) cells, the
inclusion of flecainide, QX-FL or NU-FL in the patch pipette was randomly assigned. Data
analysis was performed by operators blinded to the drugs under test.
In all experiments, data subjected to statistical analysis are from at least 5 independent values
and are reported as mean  SEM. The exact number of n in each dataset is given in each
instance in the corresponding figure legends. Data were tested for normality using the
D’Agostino and Pearson omnibus test and normally and non-normally distributed data were
tested using unpaired Student’s t-test or Mann-Whitney test, respectively. Data were
considered significant if P < 0.05. All statistical analysis was performed using Prism 6.0
software (GraphPad, CA, USA).
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Results
The structures of flecainide, QX-FL and NU-FL, together with the proportion of each
molecule present as cationic or neutral species at pH 7.4, are shown in Figure 1.
Previous work (Bannister et al. 2015) has established that flecainide, present at the cytosolic
face of the RyR2 channel, can enter the cytosolic vestibule of the channel and interact with
residues in the helices lining this portion of the pore-forming region (PFR). When bound,
flecainide introduces a physical and/or electrostatic barrier to the movement of cations from
the cytosolic to the luminal side of the channel. The affinity of interaction of flecainide at this
site is relatively weak and bound flecainide is destabilised by movement of cations in the
physiologically relevant, luminal to cytosolic direction. In this report, we have examined the
influence of QX-FL and NU-FL on cation translocation in individual recombinant hRyR2
channels reconstituted into planar phospholipid bilayers and compared these effects with
those of flecainide.
The single channel traces in Figure 2 (A) show that like flecainide, QX-FL and NU-FL,
present in the solution at the cytosolic face of the channel, are open channel blockers of
hRyR2 when net cation current is driven cytosolic to luminal by applying a holding potential
of +40 mV across the bilayer. The increase in occurrence of blocking events with increasing
concentration is evident in the traces themselves and is depicted in the accompanying
frequency amplitude histograms. As is the case with flecainide, QX-FL and NU-FL do not
fully occlude the hRyR2 pore and residual current continues to flow with the blocking
molecule bound. The expanded frequency amplitude histograms (Figure 2 (B)) highlight a
small but significant difference (p <0.05) in the residual current of the blocked states
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produced by flecainide (19.16 ± 0.61%) and QX-FL (17.27 ± 0.51%). For NU-FL the
residual current in the blocked state shows considerable variation.
In all cases block is manifest as a decrease in Po (Figure 3 (A)). The Kd for flecainide is 13.14
± 1.89 µM (n=6 channels). The fully charged and neutral analogues block considerably less
effectively, and their Kds cannot be calculated from this plot. More quantitative information
can be obtained from the rates of association (Kon) and dissociation (Koff) for flecainide, QX-
FL and NU-FL (n=6 channels) shown in Figures 3 (B) and (C). Whilst no significant
differences exist between the dissociation rates of the three compounds, rates of flecainide
association are significantly higher than those of either QX-FL or NU-FL (all in nM-1 s-1:
flecainide: 9.54 ± 2.20, QX-FL: 0.91 ± 0.03, NU-FL: 0.62 ± 0.05; p<0.05).
We next examined whether cytosolic QX-FL or NU-FL was able to influence the
physiologically relevant flux of cations through hRyR2. This was done by reversing the
holding potential across the bilayer to -40 mV and so driving net K+ current in the luminal to
the cytosolic direction (Figure 4). These experiments demonstrate that, as previously shown
for flecainide (Bannister et al. 2015), neither cytosolic QX-FL nor NU-FL is able to influence
the physiologically relevant, luminal to cytosolic flux of cations through the hRyR2 channel.
We have extended this investigation by monitoring the effects of varying holding potential on
the ability of all three forms of flecainide to block K+ flux through the open hRyR2 channel.
Figure 5 (A) shows the variation in block caused by 50 μM cytosolic flecainide, QX-FL and
NU-FL at holding potentials between  70 mV. Under these conditions the probability of
block for both flecainide and QX-FL is dependent on potential; being more pronounced as the
potential is made increasingly positive. Voltage dependence of block by the predominantly
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neutral NU-FL at 50 μM is very weak, but is more pronounced when its association rate is
increased on raising the concentration to 200 μM (Figure 5 (A)). These data confirm that for
all three compounds block is observed only when net K+ flux is in the cytosolic to luminal
direction. No block is seen at negative holding potentials when net flux is luminal to cytosolic.
In all cases both rates of blocker association (Figure 5 (B)) and dissociation (Figure 5 (C)) are
influenced by trans-membrane potential.
These characterizations establish that both QX-FL and NU-FL, present in the solution at the
cytosolic face of hRyR2 are open channel blockers of the non-physiological flux of cations
but are significantly less effective than flecainide. None of the compounds block the
physiologically relevant flux of cations when present at the cytosolic face of the channel.
Figure 6 shows the effect of adding each of the blockers to the luminal face of the channel.
Under these conditions no blocking events are seen either at a holding potential of -40 mV,
when net cation flux is in the physiologically relevant luminal to cytosolic direction, or
initially at +40 mV when net flux is cytosolic to luminal (top panel). These experiments
demonstrate that no sites of interaction for flecainide blocking molecules are present at the
luminal side of the hRyR2 channel. If a net cytosolic to luminal cation flux is maintained at
+40 mV in the presence of luminal flecainide and NU-FL, brief blocking events are apparent
after several minutes and open channel block is well established after 10-20 min (lower
panel). The neutral forms of these compounds are able to cross the membrane, passing into
the solution in the cytosolic chamber and re-equilibrate here with the cationic form (see
Figure 1). Only then are the cationic species able to access the cytosolic vestibule of the
channel and partially block cytosolic to luminal cation flux. Luminal fully charged QX-FL
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can not cross the bilayer or pass through hRyR2 and, as a consequence, has no access to
blocking sites in the cytosolic vestibule.
The data presented to this point indicate that neither QX-FL nor NU-FL are as effective as
blockers of cytosolic to luminal K+ flux through the RyR2 channel as flecainide and,
consequently, would be unable to inhibit any contribution that this current might make to
RyR2-mediated charge compensation during Ca2+ release from the SR. Previous
investigations have established that, when present in the cytosol, both flecainide and QX-FL
are potent use-dependent blockers of sarcolemmal Na+ channels (Liu et al. 2003). Their very
different ability to block a potential charge compensating current through RyR2 means that
intracellular QX-FL is a powerful tool for establishing the relative contribution of Na+
channel block, and a potential action on RyR2, to flecainide’s inhibition of inappropriate
RyR2-mediated Ca2+ release in CPVT.
Towards this end we examined the effects of intracellular flecainide and its derivatives on
Ca2+ regulation in rat ventricular myocytes. To measure Ca2+ sparks intracellular buffering
was adjusted with EGTA to prevent the occurrence of Ca2+ waves. Lowering the Ca2+-
buffering capacity of the internal solution to 0.4 mM EGTA gave optimal conditions for
selectively targeting SR-mediated spark activity. Before Ca2+ spark measurement, myocytes
from the control and drug treated group were voltage-clamp stimulated for 30 sec at 3Hz
from a resting membrane potential to +30 mV in order to bringthe SR [Ca2+] to a steady-state
level. Intracellular diastolic [Ca2+] was calculated to be 77 nM. The effects of intracellular
application of flecainide, QX-FL and NU-FL on Ca2+ spark parameters are shown in Figure 7.
A 3D topological view of the spatial and temporal properties of Ca2+ sparks under voltage-
clamp stimulation is presented in Figure 7 (A). Flecainide and QX-FL were equally effective
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at reducing the frequency of Ca2+ sparks (Figure 7 (B)), while NU-FL had no effect on this
parameter. None of the compounds had a significant effect on spark amplitude (Figure 7 (C))
or mass (Figure 7 (D)). A detailed analysis of the influence of flecainide, QX-FL and NU-FL
on Ca2+ spark parameters is presented in a table in the Supplemental Material.
Discussion
Flecainide is a well-characterized use-dependent blocker (UDB) of Na+ channels (Liu et al.
2002; Liu et al. 2003). More recently flecainide has emerged as an effective therapeutic agent
for the treatment of CPVT both in combination with conventional  blockade (van der Werf
et al. 2011; van der Werf et al. 2012; Watanabe et al. 2013) and as a mono-therapy (Padfield
et al. 2016; Napolitano 2016).
Given its effectiveness as a Na+ channel blocker, it is difficult to envisage a therapeutic action
for flecainide that does not involve this target, however its primary action in CPVT is
considered (or has been suggested) to be on RyR2, with the proposition that flecainide blocks
the open channel and hence reduces, or prevents, inappropriate RyR2-mediated release of
Ca2+ from the SR (Hilliard et al. 2010).
However, recent work from our group (Bannister et al. 2015) has established that while
flecainide does interact, with relatively low affinity, with a site within the cytosolic cavity of
RyR2, and when bound can reduce the flux of monovalent cations in the cytosolic to SR
luminal direction through the channel; bound flecainide is displaced by cation flux in the
luminal to cytosol direction (i.e. the flux corresponding to the physiologically relevant
movement of Ca2+ from the SR to the cytosol, to initiate contraction during excitation-
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contraction coupling). Given this, the only feasible mechanism by which direct interaction of
flecainide with RyR2 could influence SR Ca2+ release is by reducing a monovalent cation,
charge compensating, counter-current through the channel (Bannister et al. 2015).
While casting doubt on the therapeutic significance of a direct action of flecainide on RyR2,
this observation does raise an important mechanistic issue. In the cardiac myocyte Na+
channels and RyR2 are located in different membrane systems, however their orientation, the
direction of physiologically relevant cation flux through the channels, the access route of
flecainide to its binding site and the location of the binding site, are all equivalent (Figure
8A). Given this equivalence, why then is flecainide a therapeutically relevant blocker of the
Na+ channel but not of RyR2?
The factors governing flecainide’s blocking interaction in the Na+ channel cytosolic cavity
were revealed by studying the blocking efficiency of flecainide derivatives with similar three-
dimensional structure but differing net charge (Figure 1)(Liu et al. 2003). We have used these
derivatives in the current investigation to gain new insights into the contribution of structural
and charge characteristics to the ability of flecainide to interact with RyR2 and to block a
potential charge compensating, cytosolic to luminal, monovalent cation flux.
Our data demonstrate that (although with dramatically lower affinity), as is the case for
flecainide, both QX-FL and NU-FL are concentration- and voltage-dependent blockers of a
potential charge compensating, monovalent cation flux through RyR2. All three ligands
interact within the cytosolic vestibule of the channel pore and gain access to this site from the
cytosolic side of the channel. When bound, the ligands do not fully-occlude the RyR2 pore,
rather, they create a steric and/or electrostatic barrier that limits cytosolic to luminal cation
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flux. The different residual currents seen with the blocking molecules bound indicate that the
magnitude of the barrier is dependent upon characteristics of the blocking molecule.
Detailed investigation of the effects of flecainide, QX-FL and NU-FL on Na+ channels
established that the efficacy of these ligands as blockers was determined, primarily, by net
charge rather than the structural features of the molecule (Liu et al. 2003). The data
presented here establish that this is not the mechanism that governs the blocking interaction
with RyR2. Under the experimental conditions used in our experiments, the proportion of
flecainide and QX-FL molecules that will be positively charged is essentially the same (99%
for flecainide and 100% for QX-FL). However QX-FL is a dramatically less effective blocker
of the potential charge compensating, cytosolic to luminal, flux of K+ through RyR2. This
observation demonstrates that factors other than net charge contribute to the ability of
flecainide to block cytosolic to luminal cation flux in RyR2. This conclusion is strengthened
by the observation that NU-FL (~10% cationic) is only slightly less efficient as a blocker of
RyR2 than QX-FL. No significant difference exists in the rate of dissociation of the three
blocking molecules from RyR2 and the variation in blocking efficiency of flecainide and its
derivatives arises from differences in their rates of association with the channel, with this rate,
in comparison with flecainide, approximately 10 fold and 15 fold lower for QX-FL and NU-
FL respectively. What characteristics of the molecules underlie this variation?
Previous work from our group has established that a wide range of large mono- and
polyvalent cations are concentration- and voltage-dependent blockers of cytosolic to luminal
cation flux through the open RyR2 channel (Tinker, Lindsay and Williams 1992b; Tinker,
Lindsay and Williams 1992a; Tinker and Williams 1993; Mead and Williams 2004; Mason et
al. 2012). Given this, it is logical to conclude that the open channel block reported here arises
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from the interaction of the cationic component of flecainide, QX-FL and NU-FL, with a site
within the cytosolic vestibule of RyR2. Therefore the difference in the absolute concentration
of cationic species in flecainide and NU-FL could be a contributing factor to the differing
abilities of these molecules to block RyR2. However, as the absolute concentrations of
cationic species in flecainide and QX-FL are essentially the same, other molecular
characteristics must contribute to the difference in the efficacy of these blockers.
In Na+ channels, local anesthetic antiarrhythmics, including flecainide, are stabilized within
the cytosolic vestibule by interactions with two aromatic residues separated by two turns on
the S6 pore-lining helix of repeat IV (Ragsdale et al. 1994; Ragsdale et al. 1996), and high
affinity binding is believed to involve cation-pi interactions between these aromatic residues
and amine groups of the blockers (Ahern et al. 2008). In RyR2 cation-pi interactions are
unlikely to underpin flecainide block since there are no aromatic residues in the proposed
binding region (the lower cytosolic vestibule of the channel) (Bannister et al. 2015). The
variation in blocking efficiency of flecainide and its derivatives suggests that the capacity of
the molecules to act as hydrogen bond acceptors or donors may be important in determining
their ability to bind in the RyR2 cytosolic cavity. Considering functional group differences,
the secondary amine flecainide can form two hydrogen bonds with the piperidine ring as its
conjugate acid, whereas NU-FL, a tertiary amine, can form only one. QX-FL, as a quaternary
compound, has no hydrogen bonding capacity in that region. Small structural differences
between flecainide, QX-FL and NU-FL (Figure 1) may also contribute to the likelihood of
interaction with RyR2. The bonding around the ring nitrogen in QX-FL and the cationic
forms of flecainide and NU-FL is not identical, and steric encumbrance around this atom may
result in reduced efficacy.
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Crucially, the consequence of flecainide interaction with the Na+ channel differs from that
with RyR2 in one very important way: although access to the flecainide binding site in Na+
channels requires the channel to be open, once bound, flecainide interacts preferentially with
the inactivated closed conformation of the channel, prolonging this state, thereby limiting the
channel’s availability for further activation (Liu et al. 2002). This limited egress of flecainide
from the Na+ channel facilitates the accumulation of block (or use-dependence) with
repetitive depolarizations of the cell (Liu et al. 2003). Therefore, in Na+ channels flecainide
does not merely act as a blocking substrate, but also effectively alters the gating of the
channel. RyR2 gating involves transitions between open or closed states; unlike the Na+
channel, the channel does not need to pass through an inactivated state, so in RyR2 flecainide
acts simply as an open channel partial blocker of cytosolic to luminal cation flux and use-
dependent block does not occur. The absence of an effect on gating, and the low affinity with
which flecainide binds within the cytosolic vestibule of RyR2, means that bound flecainide is
readily displaced by the physiologically relevant, luminal to cytosolic, flux of Ca2+ from the
SR during E-C coupling (Bannister et al. 2015). The mechanisms governing flecainide
interaction in Na+ and RyR2 channels are summarised in Figure 8 (B) and (C) respectively.
The data presented here and in Bannister et al (2015) establish that the only way in which
flecainide could act directly on RyR2 to inhibit Ca2+ release would be via a very small
reduction of an RyR2-mediated counter current. The demonstration that QX-FL is essentially
without effect on this current identifies this ligand as a tool with which to examine if counter
current inhibition contributes, in any way, to flecainide’s therapeutic action in CPVT.
Intracellular QX-FL and flecainide are both highly effective blockers of the sarcolemmal Na+
channel (Liu et al. 2003, Supplementary Figure 2), while even the small, potential, effect of
flecainide on RyR2-mediated counter current will be absent with intracellular QX-FL.
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Consistent with the conclusion that the action of flecainide on Ca2+ handling in cardiac
myocytes results solely from inhibition of the Na+ channel, intracellular flecainide and QX-
FL have equivalent effects on Ca2+ sparks. NU-FL, a considerably less efficient Na+ channel
blocker, has no effect on Ca2+ handling.
In intact cardiomyocytes whole-cell patch clamp-mediated intracellular application of 5 μM
flecainide and QX-FL, but not NU-FL, decreased spark frequency with no effect on spark
amplitude and mass (Figure 7 and Supplementary Table). These data suggest that it is the
charged form of flecainide that mediates this effect and corroborate and extend previous
findings that flecainide suppresses Ca2+ spark frequency (Sikkel et al 2013a). Taken together
with the present investigations that confirmed a lack of effect of these concentrations of
flecainide and QX-FL on the physiologically relevant luminal-to-cytoplasmic ion flux
through RyR2 and, in the case of QX-FL, any inhibition of a potential charge compensating
cytosolic-to-luminal K+ flux (Figures 4-6), these data support the conclusion that the only
mode of action underpinning flecainide’s efficacy in CPVT patients is the inhibition of INa
(Sikkel et al 2013a; Bannister et al. 2015; Liu et al. 2011). In addition, our demonstration that
QX-FL is a Na+ channel specific ligand, yet has identical effects to flecainide on Ca2+ spark
parameters (Supplementary Table) reinforces this conclusion.
Sikkel et al. (2013a) showed that the flecainide-mediated reduction in Na+ influx into
the cardiomyocyte can, via the enhancement of Ca2+ efflux through the Na+/ Ca2+ exchanger
(NCX), decrease [Ca2+]i in the vicinity of the RyR2 and thus reduce the frequency of
spontaneous SR Ca2+ release events. These authors also showed that this was a class effect of
INa blockers and moreover, flecainide caused no reduction in SR Ca2+ leak when INa was
eliminated by altering the holding potential via voltage clamp. By amalgamating these
findings with their existent hypothesis that flecainide’s mechanism of action was dependent
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on direct effects on INa and through direct interaction with RyR2, Steele and co-workers
proposed a ‘triple mode’ of flecainide action namely via its direct effects on INa and RyR2
and an indirect effect on NCX (Steele et al. 2013). The present investigations, which
corroborates previous work by Bannister and colleagues (Bannister et al. 2015), present the
unequivocal demonstration that flecainide does not have a direct effect on luminal-to-
cytosolic Ca2+ flux through RyR2 and fundamentally challenges reports that flecainide
efficacy in RyR2- and CSQ-mutation linked CPVT patients is due to an additive effect of INa
inhibition and a direct block of RyR2 (Watanabe et al. 2009; Hilliard et al. 2010; Lee et al.
2012; Hwang et al. 2011; Galimberti and Knollmann 2011). Further corroborating the lack of
direct effect of flecainide on RyR2-mediated Ca2+ release, Supplementary Figure 3 shows the
persistence of RyR2-dependent spontaneous Ca2+ release events in Nav1.5-null human
embryonic kidney (HEK) in the presence of flecainide (5 µM).
The contention that flecainide is less effective in inhibiting RyR2 under circumstances
that promote higher spark frequency (i.e. conditions of Ca2+ overload) (Steele et al. 2013;
Sikkel et al 2013b) is not applicable here since the present studies in intact control cells are
characterised by comparable Ca2+ spark frequencies to those reported by Hilliard et al (≈ 1.5
versus 0.8, respectively) suggesting similar SR Ca2+ loading. Adding to the confusion, the
same group have recently described the increased potency of flecainide in suppressing
intracellular Ca2+ waves in permeabilized cardiomyocytes following manoeuvres that
increase Ca2+ spark frequencies (Savio-Galimberti and Knollmann 2015).
We have established that the principal action of flecainide in CPVT is not via a direct
interaction with RyR2. Our data support a model of flecainide action in which Na+-dependent
modulation of intracellular Ca2+ handling attenuates RyR2 dysfunction in CPVT. These
findings are crucial as they will contribute to the rational design of improved therapies, and
prevent the clinical misuse of flecainide in the treatment of phenotypically similar but
mechanistically distinct arrhythmias.
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Acknowledgements
This work was supported by Project Grant PG14/34/30835 from the British Heart Foundation.
Author contributions
A.J.W., M.L.B., N.L.T., A.A.L. and C.H.G. designed the study; M.L.B. and N.L.T.
performed single channel experiments; A.A.L. performed myocyte Ca2+ imaging
experiments; S.C. and C.H.G. created the H-Flp/Nav1.5 cells; S.A.M. characterised Na+
channel function; C.d.P., A.T.M., D. N-H., H.O. and M.C.B. synthesised NU-FL and QX-FL;
M.L.B., N.L.T., A.A.L., S.A.M, K.T.M. and C.H.G. analysed the data; M.L.B., N.L.T.,
A.J.W., C.H.G., A.A.L., K.T.M., S.A.M. and M.C.B. wrote the paper.
Conflicts of interest
The authors declare no conflict of interest.
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Figure Legends
Figure 1 Chemical structures of flecainide, QX-FL and NU-FL. At pH 7.4, flecainide (pKa
9.3) is >99% charged; NU-FL (pKa 6.4) is >90% neutral. QX-FL is permanently charged.
Figure 2 Open channel block of voltage driven cytosolic to luminal cation flux through
hRyR2. (A) Representative single channel traces recorded at + 40 mV in symmetrical 610
mM KCl. Openings are downwards from the closed level (black line). Channels are fully
activated with 20 μM EMD 41000. The increase in blocking events (marked with a dotted
line) with increasing concentration of flecainide, QX-FL or NU-FL is depicted in the
frequency amplitude histograms and manifests as a decrease in Po and increase in Pb (B)
Expanded frequency amplitude histograms demonstrating the nature of the blocked states,
tables above show the mean residual current (R.C.), Pb and Tb achieved with 50 μM of each
drug. n is given in each instance.
Figure 3 QX-FL and NU-FL are less potent blockers of the cytosolic to luminal current
through hRyR2 compared to flecainide. (A) Decrease in Po at +40mV observed with
increasing concentrations of flecainide, QX-FL or NU-FL with corresponding (B) rates of
association (Kon, fitted through the origin) and (C) dissociation (Koff). All data points are from
n=6 separate experiments.
Figure 4 Neither flecainide, nor its derivatives block voltage driven luminal to cytosolic
cation flux through hRyR2. Representative single channel traces, recorded at – 40 mV
(luminal to cytosolic current) in the presence of cytosolic ligand (activated with 20 μM
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EMD41000). Openings are upwards from the closed level (black line); no blocking events or
significant changes in Po were observed (n = 6).
Figure 5 Voltage dependence of block. (A) Plot of Po vs voltage for hRyR2 channels in the
presence of 50 μM cytosolic blocker (flecainide n = 11, QX-FL n=8, NU-FL n = 5)
determined at holding potentials between ± 70 mV. A higher concentration of NU-FL (200
μM, n = 5) is required to see voltage dependence. Dose normalised rates of (B) association
(Kon) and (C) dissociation (Koff) are both voltage-dependent for flecainide, QX-FL (each at 50
μM) and NU-FL (200 μM).
Figure 6 Addition of flecainide or its derivatives to the luminal face of channel.
Representative traces of single RyR2 channels recorded at + 40 mV before and after the
addition of high concentrations of blocker (top panel). QX-FL does not cross the bilayer and
cannot access the channel cytosolic vestibule. Flecainide and NU-FL are able to equilibrate
across the membrane and block the cytosolic to luminal current only (lower panel). Data were
obtained from n=3 independent experiments and were not subjected to statistical analysis.
Figure 7 Effects of intracellular application of flecainide and its analogues on Ca2+ spark
parameters. (A) 3D topological view of the spatial and temporal properties of Ca2+ sparks
under voltage-clamp stimulation. Cells were dialyzed via patch pipette (10-12 min) with
either vehicle control solution containing Fluo-3-5K+ salt, or with flecainide, QX-FL or NU-
FL (5 μM). Myocytes were stimulated via depolarization from -80 to +30 mV (2Hz, 30s) and
spontaneous sparks recorded during a subsequent 20s quiescent period. (B-D): The effect of
intracellular dialysis of flecainide, QX-FL or NU-FL on mean Ca2+ spark frequency (B),
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