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Low pH depolarizes the voltage dependence of voltage-gated sodium (Na(V)) channel activation and fast inactivation. A complete description of Na(V) channel proton modulation, however, has not been reported. The majority of Na(V) channel proton modulation studies have been completed in intact tissue. Additionally, several Na(V) channel isoforms are expressed in cardiac tissue. Characterizing the proton modulation of the cardiac Na(V) channel, Na(V)1.5, will thus help define its contribution to ischemic arrhythmogenesis, where extracellular pH drops from pH 7.4 to as low as pH 6.0 within ~10 min of its onset. We expressed the human variant of Na(V)1.5 with and without the modulating β(1) subunit in Xenopus oocytes. Lowering extracellular pH from 7.4 to 6.0 affected a range of biophysical gating properties heretofore unreported. Specifically, acidic pH destabilized the fast-inactivated and slow-inactivated states, and elevated persistent I(Na). These data were incorporated into a ventricular action potential model that displayed a reduced maximum rate of depolarization as well as disparate increases in epicardial, mid-myocardial, and endocardial action potential durations, indicative of an increased heterogeneity of repolarization. Portions of these data were previously reported in abstract form.

Ranolazine is a clinically approved anti-anginal drug with potential antiarrhythmic, antiepileptic, and analgesic applications. The therapeutic effects of ranolazine are dependent on its ability to preferentially inhibit persistent currents in a variety of voltage-gated sodium channels. Extracellular acidosis, as occurs during ischemic events, may alter the interaction between ranolazine and the channel. In this study, we performed whole-cell patch clamp experiments with extracellular solution titrated to pH 7.4 or pH 6.0 using HEK cells expressing NaV1.5 and CHO cells expressing NaV1.2. We found that ranolazine modulates these sodium channels with onset/recovery kinetics and voltage-dependence resembling slow inactivation. In this way, ranolazine increases use-dependent inactivation of the channel and decreases window currents. At pH 6.0, ranolazine interaction with the sodium channel is slowed approximately 4-5 fold in both NaV1.2 and NaV1.5. Despite the slowed kinetics, ranolazine remains effective at steady-state during acidic conditions. At low extracellular pH ranolazine rescues the voltage-dependence of slow inactivation at a therapeutically relevant concentration (10µM). Our results suggest that, at pH 6.0, ranolazine compensates for proton-induced impaired slow inactivation and remains effective at reducing persistent currents.

Ranolazine is an anti-anginal drug previously shown to block persistent currents of the cardiac voltage gated sodium channel, NaV1.5. The effects of ranolazine, however, have not yet been described in all sodium channel isoforms. We studied the effects of ranolazine on the neuronal sodium channel isoform, NaV1.2, and its modulation by extracellular protons. Ionic currents were measured from Chinese Hamster Ovary (CHO) cells expressing the α-subunit of NaV1.2, using whole cell patch clamp techniques. Voltage protocols were run with extracellular solutions of pH 7.4 and pH 6.0 before and after the addition of ranolazine. The addition of 100 μM ranolazine at pH 7.4 led to a significant decrease in late sodium current, faster rate of open state fast inactivation, and slower recovery from inactivation. Similar trends were seen in preliminary experiments at 10 μM and 30 μM ranolazine. In addition, we observed a tonic block of peak current and an increase in total use-dependent block. Many of these effects were different at low pH. Low pH led to a significant depolarizing shift of the conductance curve, and slowed the onset of fast inactivation. Adding ranolazine at pH 6.0 significantly decreased the rate of fast inactivation recovery, and increased the total use-dependent block and rate of open state inactivation. Although directions of the effects were unchanged, the magnitude of these effects was significantly different between the two pH values. Our results suggest that ranolazine stabilizes fast inactivation at both pH 7.4, and pH 6.0. It is possible that ranolazine and protons may compete for a common binding site or indirectly interact leading to decreased ranolazine efficacy at low pH. (Supported by a research grant from Gilead Sciences, Inc. and an NSERC Discovery Grant to PCR, and an NSERC URSA to CHP.)

Brugada Syndrome (BrS) is an inherited cardiac arrhythmia implicated in SIDS and sudden death in young men. BrS is caused by loss-of-function mutations in the cardiac voltage-gated sodium channel NaV1.5. The decrease in sodium current can lead to electrical abnormalities in the ventricular action potential that can degenerate into ventricular tachycardia. The ECG phenotype and electrical abnormalities associated with BrS are not always present and are often unmasked clinically with sodium channel blockers. Paradoxically, BrS associated arrhythmias manifest during and after exercise as well as in sleeping infants. We hypothesize that one trigger for arrhythmias in BrS may be extracellular acidosis, which can occur during both exercise and sleep apnea. We used whole-cell patch clamp to characterize 3 BrS mutants, R376H, R1193Q, and E1784K, at pH 7.4 and pH 6.0. At pH 7.4 all mutants have reduced current compared to WT channels and there are changes in channel activation and fast inactivation. At pH 6.0, E1784K showed the largest proton-dependent effect with a 20 mV depolarizing shift in the midpoint voltage of activation compared to the 12 mV shift in WT. All 3 mutants showed decreased function at pH 6.0 although the magnitude of the shifts was not always different from WT. Overall, extracellular acidosis would be expected to challenge already decreased sodium currents, particularly in the E1784K mutant. Therefore, acidosis may be one of the triggers for arrhythmia in BrS patients.

EAG, ERG and ELK are members of the Ether-a-go-go voltage-gated potassium channel family. Each channel possesses a C-terminal domain that is homologous to the cyclic nucleotide-binding domain (CNBD) of CNG/HCN channels but insensitive to cyclic nucleotides. X-ray crystallography of EAG suggests the side chains of two residues, Y699 and L701, interact in a manner analogous to the two moieties of a cyclic nucleotide in the binding pocket of bona fide CNBDs. Using two-electrode voltage clamp of channel proteins expressed in Xenopus oocytes, we found that alanine substitutions of Y699 and L701 (“AA”) dramatically slowed activation and shifted the g-V curve by +15 mV. Mutagenesis of both residues were required to elicit an effect, as if the “apo” configuration required the loss of both side chains. Thus, the intrinsic ligand of the CNB homology domain (CNBhD) reduces the stability of the closed state relative to the open state. To determine whether the AA mutant phenotype represents a loss of function, we deleted the CNBhD. In contrast to the AA mutant, the deletion gave rise to faster activation kinetics but a more dramatic g-V shift of +40 mV. This finding indicates that, unlike HCN channel, in which the unliganded CNBD mimics the CNBD deletion, the AA mutant phenotype is not a loss of function of the CNBhD. Instead, both the intrinsically-liganded and apo conformations communicate with the gating machinery. The current properties can be recapitulated by a 7-state allosteric model, in which the AA mutant alters multiple transitions during channel gating whereas CNBhD deletion affects only the last opening step. We conclude that the EAG1 CNBhD serves a function mechanistically distinct from those of the corresponding domains in channels gated by cyclic nucleotides despite extensive sequence and structural homology.