Ranolazine inhibition of hERG potassium channels: Drug–pore interactions and reduced potency against inactivation mutants

The antianginal drug ranolazine, which combines inhibitory actions on rapid and sustained sodium currents with inhibition of the hERG/IKr potassium channel, shows promise as an antiarrhythmic agent. This study investigated the structural basis of hERG block by ranolazine, with lidocaine used as a low potency, structurally similar comparator. Recordings of hERG current (IhERG) were made from cell lines expressing wild-type (WT) or mutant hERG channels. Docking simulations were performed using homology models built on MthK and KvAP templates. In conventional voltage clamp, ranolazine inhibited IhERG with an IC50 of 8.03 μM; peak IhERG during ventricular action potential clamp was inhibited ~ 62% at 10 μM. The IC50 values for ranolazine inhibition of the S620T inactivation deficient and N588K attenuated inactivation mutants were respectively ~ 73-fold and ~ 15-fold that for WT IhERG. Mutations near the bottom of the selectivity filter (V625A, S624A, T623A) exhibited IC50s between ~ 8 and 19-fold that for WT IhERG, whilst the Y652A and F656A S6 mutations had IC50s ~ 22-fold and 53-fold WT controls. Low potency lidocaine was comparatively insensitive to both pore helix and S6 mutations, but was sensitive to direction of K+ flux and particularly to loss of inactivation, with an IC50 for S620T-hERG ~ 49-fold that for WT IhERG. Docking simulations indicated that the larger size of ranolazine gives it potential for a greater range of interactions with hERG pore side chains compared to lidocaine, in particular enabling interaction of its two aromatic groups with side chains of both Y652 and F656. The N588K mutation is responsible for the SQT1 variant of short QT syndrome and our data suggest that ranolazine is unlikely to be effective against IKr/hERG in SQT1 patients.


Maintenance of mammalian cell lines and cell transfection
HEK 293 cells stably expressing wild-type (WT) hERG (provided by Professor Craig January [3]) or the Y652A and F656A mutants [4] were passaged using enzyme free cell dissociation solution (Millipore, Watford, UK) and a non-enzymatic agent (Enzyme Free, Chemicon® International) and were maintained as described previously [4][5][6][7]. For transient transfection, cells were maintained as previously described [8] and were transiently transfected with cDNA plasmids (T623A, S624A and V625A hERG) using either Lipofectamine 2000 or LTX (Invitrogen, Paisley, UK) according to the manufacture's instructions. The N588K and S620T mutants were transiently transfected into Chinese Hamster Ovary cells using previously described methods. Green Fluorescent Protein (GFP, in a pCMX vector, was donated by Dr. Jeremy Tavare) [9,10] or CD8 (in pIRES, donated by Dr I Baró, University of Nantes, France) [7,8] were used as markers of successful transfection. Successfully transfected cells were identified using either green fluorescence (with excitation at 490 nm and emission at 510 nm) or Dynabeads® (Invitrogen, Paisley, UK). After transfection, the cells were incubated at 37 o C for a minimum of 1 day prior to any electrophysiological study.

Electrophysiological recordings
Glass coverslips onto which cells had been plated were placed in a chamber mounted on the microscope and the cells were continuously superfused at 37 ± clamp commands were generated as described previously [7,8]. Data were recorded via a Digidata 1200B interface (Axon Instruments, USA) and stored on the hard-disk of a Viglen computer. A data digitization rate of 10 kHz was used during all protocols and a bandwidth of 2 kHz was set on the amplifier.

Data analysis
The fractional block of hERG I tails was determined using an equation of the form: Where y is fractional block at a given concentration of Drug ([Drug]), IC 50 is the Drug concentration producing half-maximal inhibition of I hERG and h is the Hill coefficient for the fit to the plotted data.
Data are presented as mean ± S.E.M. Statistical comparisons were made using paired or unpaired t tests or one/two-way analysis of variance (ANOVA).

Molecular modelling
The hERG pore model built onto the crystal structure of the bacterial K + channel MthK (PDB: 1LNQ; [12]) is described in detail elsewhere [7,13]. In previous docking studies we found that a modified KvAP model ("Farid model") [14] selected drug binding poses similar to those found for the MthK model. [7]. We have found that homology models built on a putative C-type inactivated state structures of the KcsA bacterial channel do not accord well with experimental data on drugbinding [15], whilst a MthK based model generally shows good concordance [15]. By contrast, although we have previously constructed a full hERG open state homology model based on a mammalian (Kv1.2/2.1 paddle chimera) template [16], this has a proline (PVP) motif that overlaps S6 residues of the canonical drug binding site (see Figure 1 in [16]). The structure of the hERG S6 helix is unlikely to be equivalent to that of the mammalian templates in this part of the pore and consequently we chose to employ bacterial channel template based models established to show good agreement with experimental drug binding data [14,15]. Docking analyses were run in parallel using both the MthK and Farid models in order to minimize model-dependence in the docking outputs and interpretations. In all models the selectivity filter contained K + ions in the S1 and S3 positions of the selectivity filter. K + ions were introduced by superimposing the selectivity filter of the high resolution KvAP structure using Insight II (Accelrys), and transferring K + ions into the appropriate sites of the models.
Since the crystal structures upon which the hERG pore homology models were constructed are static, we introduced pore model flexibility by allowing free side chain bond rotation for residues in or near the bottom of the selectivity filter (T623, S624 and V625) and for residues that project from the S6 helices towards the pore cavity (Y652, F656, S660). Free conformational sampling around all rotatable bonds in the drug molecules was allowed. In each docking run 60,000 generations of the Genetic Algorithm were run; to increase sampling of configurational space each docking run of each drug into each model was repeated 240 times using different starting configurations with the drug repositioned and reoriented in the pore cavity.
In a set of runs made to explore possible binding of drugs near S620, the drug binding cavity was redefined to include S620 and all residues within 10 Å and an appropriate set of new residue beside chain bond rotations (including S620) was sampled. Since Flexidock was unable to find low energy score docked poses involving direct interaction of drugs with S620, these results are not shown here.

Supplementary Results
. Figure S2: Block of WT I hERG by lidocaine (A) Effects of lidocaine studied using the same standard I hERG pharmacology protocol (described in the main text, and shown above as the lower trace in A) as used for ranolazine. Upper traces in A show WT I hERG elicited in Control solution and in the presence of 100 M lidocaine.
(B) Upper trace shows voltage protocol used to elicit WT I hERG in 94 mM [K + ] e . Boxed area shows repolarizing phase of protocol from +20 to -120 mV. Lower trace shows WT I hERG records on repolarization to -120 mV in Control and in 100 M lidocaine. Horizontal arrows in A and B denote zero current. The brief (50 ms) steps to -40 mV and -120 mV A B C A B C in A and B respectively elicited reference (nominally hERG-free) current against which peak outward/inward I hERG was measured. (C) shows concentration-response relations for inhibition of WT I hERG by lidocaine in 4 mM [K + ] e (filled squares) and in 94 mM [K + ] e (open circles). Data were fitted with a Hill-equation. Note that error bars for some points are small and are obscured by the symbols (n  5 cells per data-point). Figure S2 shows results from similar experiments performed using lidocaine to those shown in Figure 1 (main paper text) on ranolazine. Figure S2A shows representative control and lidocaine (100 M) traces in normal (4mM) [K + ] e . Four lidocaine concentrations were studied, mean fractional block for each concentration then calculated and a concentration-response relation constructed as shown in Figure S2C (filled squares). The derived IC 50 was 141.77 ± 9.81 µM (h=0.98 ± 0.07). Figure

Effect of ranolazine on voltage-dependence of WT I hERG
The voltage-dependence of I hERG inhibition by ranolazine was determined using the protocol shown in lower panels of Figures S3A and B (comprised of 2s steps to different test potentials, followed by repolarization to -40 mV to elicit current tails; I tail ). Successive command pulses were applied at 12-second intervals. The protocol was applied in control and during superfusion with ranolazine-containing solutions. Figure S3 A and B show example WT I hERG current traces recorded from the same cell in the absence (A) and presence (B) of 10 µM ranolazine. I hERG amplitude both during depolarizing steps and on repolarization was reduced when cells were exposed to 10 µM ranolazine ( Figure S3B). In order to determine ranolazine inhibition of I hERG at different voltages, I hERG amplitude at the end of the depolarization step was measured and the I-V relationship was constructed as shown in Figure S3C. For each cell and each condition (control, drug) current amplitude was normalized to the maximal control current during the protocol. The I-V relationship showed a characteristic bell-shaped appearance both in control and in the presence of 10 µM ranolazine. I hERG amplitude was smaller in the presence of ranolazine between voltages from -20 to +60 mV (indicated by symbols above points on Figure 6.4 C, two-way ANOVA followed by a Bonferroni post-test, n= 6 cells).

Figure S3
Effect of ranolazine on the voltage-dependence of I hERG (A, B). Representative families of I hERG in the absence (A) and presence (B) of 10 µM ranolazine elicited by the voltage protocol shown in lower panels (same protocol as shown in Figure 3.3). Some current traces are omitted for clarity of display. (C) I-V relations for end-pulse current with and without 10 µM ranolazine (n= 6 cells). Data were normalised to the maximal I hERG current during the protocol and then plotted against test potentials. (D) Steady-state activation plots for I hERG derived from tail current measurements on repolarization to -40 mV in the absence and presence of 10 µM ranolazine. The I tails were normalised to the maximal tail current recorded during the protocol and then plotted against test potential. (E) Plot of mean (±SEM) fractional block of I hERG against test voltage for end-pulse current (n = 6 cells). (F) Plot of mean (±SEM) fractional block of I hERG tails against test voltage (n = 6 cells). Note that error bars for some of the points are small and are obscured by the symbols. "*" denotes statistical significance of p<0.05; "***" denotes statistical significance of p<0.001; (2way ANOVA followed by a Bonferroni post-test).
The voltage-dependence of I hERG activation was evaluated by plotting normalised I-V relations for the hERG I tails (with I tail at each voltage in each condition normalized to the maximal I tail amplitude during the protocol) and is shown in Figure S3D: for currents in both control and ranolazine, I tail amplitude increased with depolarization to more positive potentials, reaching a plateau at ~ +10 mV. The data were fitted with a standard Boltzmann function to give a V 0.5 of -14.80 ±1.39 mV in control (k= 5.72 ±0.32 mV) and a V 0.5 of -18.95 ±1.87 mV (k= 5.53 ±0.14 mV) with 10 µM of ranolazine (p< 0.05, paired t-test; n= 6 cells). Thus the voltage-dependence of I hERG activation was negatively shifted by ranolazine. Voltage-dependence of current inhibition by ranolazine was also assessed and is shown in Figure S3 E and F. One-way ANOVA analysis showed inhibition to be voltage-dependent for both end-pulse and tail currents (p<0.05). For the I end plots, the fractional inhibition at -40 mV was significantly smaller than at other voltages; whilst for I tail plots, those blocking values at -40 and -30 mV were significantly smaller than at other voltages. There was no significant difference in fractional block over the potential range between -20 mV and +60 mV for either end-pulse or tail current (n= 6 cells; one-way ANOVA, followed by Bonferroni post-test).
These findings are consistent with those of Rajamani and colleagues [18], who also found I hERG inhibition by ranolazine during step protocols to show voltage dependence and observed a leftward shift in voltage-dependent activation (by ~-7 mV with 30 M ranolazine; compared to ~-4 mV with 10 M ranolazine in the present study).

Effect of the S620T hERG mutation on the action of lidocaine
For comparison with ranolazine, further experiments were performed in which the effects of the S620T mutation on lidocaine block of I hERG were examined. The same voltage protocol was used as used for both the WT I hERG concentration-response data and to study effects of S620T on ranolazine block of I hERG . Figure S4A shows the effect of a high (1mM) concentration of lidocaine on S620T I hERG . Only modest I hERG inhibition was produced by this lidocaine concentration (which produced >80% inhibition of WT I hERG ). Figure S4B shows the concentration-response relation for S620T I hERG inhibition, also displayed in the same plot is the corresponding relation for WT I hERG. The derived IC 50 for lidocaine inhibition of S620T was 6880 ± 1110 µM (h= 0.51 ± 0.05), which is ~49-fold its WT control. The suppression of lidocaine block of I hERG by the F656A and Y652A mutations (main text Figures   3Bii and 3Dii) was very small, indicating that neither of these side chains is essential for block. To assess whether these observations can be understood in terms of a degeneracy/redundancy in potential binding partners for the lidocaine aromatic group we docked the drug into hERG pore models carrying either the Y652A or F656A mutation ( Figure S5). These runs showed that low energy score poses can be obtained with the tertiary amino group positioned near the pore cavity K + binding site, and the lidocaine aromatic group making interactions with either F656 or Y652 ( Figure S5). Thus it was possible for lidocaine block to be maintained in Y652A and F656A mutants because the lidocaine aromatic ring was able to make interactions with either a Y652 (in the case of F656A) or a F656 (in the case of Y652A) aromatic side chain.
In the case of ranolazine, both Y652A and F656A mutations resulted in a loss of aromatic interactions involving one of the aromatic groups on the drug ( Figure S6), consistent with the large decreases in affinity for block of each of these mutant channels (main text Figures 3Bi and 3Di).
Unlike lidocaine, interactions with both Y652 and F656 aromatic side chains are required to express full hERG block by ranolazine.   Figure S5.

Potency of hERG blockers with inactivated-state preference scales with the number of interactions between drug and hERG pore residues
We recently showed that the IC 50 values of hERG blockers with blocking affinity enhanced by inactivation was related to the number of defined interactions each drug made with amino acid residues in the MthK-based hERG pore model used here [15]. Ranolazine and lidocaine conform to this relationship ( Figure S7) supporting the interpretation that the enhanced binding of ranolazine in the hERG pore cavity compared to lidocaine results from the larger number of interactions possible with the larger drug compared to the smaller one. In this analysis the number of defined interactions between drug and hERG pore residues was averaged over the 5 lowest energy score docked outputs for each drug. Defined interactions comprise hydrogen bonds, cation- and interactions between aromatic rings, and the location of the protonated aliphatic amino group near the cavity binding site of a K + ion in high resolution structures of homologous ion channels as indicated in Figures 5, S5 and S6. The distance criteria for these interactions are defined in reference 15. Figure S7: Relationship between hERG block potency (log IC 50 ) and number of interactions with hERG pore residues for hERG blockers including ranolazine and lidocaine measured in low energy score docking outputs from the MthK model using FlexiDock. The numbered hERG blockers are: 1, cavalli-6; 2, dofetilide; 3, terfenadine; 4, E-4031; 5, cavalli-2; 6, cisapride; 7, haloperidol; 8, chloroquine; 9, S-bupivacaine. See reference 15 for further details.  These summary tables show IC 50 values for WT I hERG tails using the standard +20 mV protocol, with I tail measured at -40 mV and for measurements at -120 mV in high [K + ] e . Comparable summary data are then shown for the mutant hERG channels studied. The right-most column shows fold-changes in IC 50 with each mutant compared to their relevant WT control, or for WT measurements with WT-1