Colin H. Peters

Extracellular Proton Modulation of the Cardiac Voltage-Gated Sodium Channel, NaV1.5

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.

Acidosis Differentially Modulates Inactivation in NaV1.2, NaV1.4, and NaV1.5 Channels

NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of NaV1.2, NaV1.4, and NaV1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas NaV1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in NaV1.2 and NaV1.5 were profound. In NaV1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in NaV1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability.

Triggers for arrhythmogenesis in the Brugada and long QT 3 syndromes.

Cardiac arrhythmias are a prevalent cause of morbidity and mortality. In many cases, inheritable mutations in the genes encoding cardiac ion channels are the underlying cause of arrhythmias. Relative to other arrhythmogenic disorders, Brugada syndrome (BrS) is recently identified and not well-understood. Although most often referred to as a disease of cardiac sodium channels, familial BrS is now associated with 9 different genes. Of these genes, 4 alter sodium currents, and the most common known genetic cause remains loss-of-function mutants in the cardiac sodium channel gene SCN5A. Long QT syndrome (LQTs) has a much longer history and is associated with at least 17 genes. LQT3, which is the third most common LQTs, is due to gain-of-function mutations in SCN5A. The first sign for BrS and LQTs patients may be sudden death. The triggers for these sudden deaths include exercise, fever, ischemia, and drug use. In this paper we review the effects of acidosis and fever on BrS and LQTs, discuss Brugada phenocopy syndrome drawing from published literature, and present our own patch-clamp data from mutant channels at low pH. We show that, at low pH, there is a preferential block of peak currents and preferential increase of persistent current in a common BrS/LQTs mutant compared to wild type sodium channels. Our data complements the existing literature on the importance of environmental triggers to arrhythmias.

Introduction to Sodium Channels

Voltage-gated sodium channels (VGSCs) are present in many tissue types within the human body including both cardiac and neuronal tissues. Like other channels, VGSCs activate, deactivate, and inactivate in response to changes in membrane potential. VGSCs also have a similar structure to other channels: 24 transmembrane segments arranged into four domains that surround a central pore. The structure and electrical activity of these channels allows them to create and respond to electrical signals in the body. Because of their distribution throughout the body, VGSCs are implicated in a variety of diseases including epilepsy, cardiac arrhythmias, and neuropathic pain. As such the study of these channels is essential. This brief review will introduce sodium channel structure, physiology, and pathophysiology.

Proton Sensors in the Pore Domain of the Cardiac Voltage-Gated Sodium Channel

Background: Protons modify cardiac sodium channel function, potentially contributing to cardiac arrhythmia during and following ischemia. Results: Protons binding amino acids at the pore alter cardiac sodium channel function. Conclusion: Sodium channel pore protonation mediates proton block and destabilization of sodium channel slow inactivation. Significance: Understanding sodium channel proton modulation is necessary to understand the pathophysiology of cardiac acidosis. Protons impart isoform-specific modulation of inactivation in neuronal, skeletal muscle, and cardiac voltage-gated sodium (NaV) channels. Although the structural basis of proton block in NaV channels has been well described, the amino acid residues responsible for the changes in NaV kinetics during extracellular acidosis are as yet unknown. We expressed wild-type (WT) and two pore mutant constructs (H880Q and C373F) of the human cardiac NaV channel, NaV1.5, in Xenopus oocytes. C373F and H880Q both attenuated proton block, abolished proton modulation of use-dependent inactivation, and altered pH modulation of the steady-state and kinetic parameters of slow inactivation. Additionally, C373F significantly reduced the maximum probability of use-dependent inactivation and slow inactivation, relative to WT. H880Q also significantly reduced the maximum probability of slow inactivation and shifted the voltage dependence of activation and fast inactivation to more positive potentials, relative to WT. These data suggest that Cys-373 and His-880 in NaV1.5 are proton sensors for use-dependent and slow inactivation and have implications in isoform-specific modulation of NaV channels.

Effects of the antianginal drug, ranolazine, on the brain sodium channel NaV1.2 and its modulation by extracellular protons

Ranolazine is an antianginal drug currently approved for treatment of angina pectoris in the United States. Recent studies have focused on its effects on neuronal channels and its possible therapeutic uses in the nervous system. We characterized how ranolazine affects the brain sodium channel, NaV1.2, and how its actions are modulated by low pH. In this way, we further explore ranolazines potential as an anticonvulsant and its efficacy in conditions like those during an ischaemic stroke.

Proton-dependent inhibition of the cardiac sodium channel Nav1.5 by ranolazine

Ranolazine is clinically approved for treatment of angina pectoris and is a potential candidate for antiarrhythmic, antiepileptic, and analgesic applications. These therapeutic effects of ranolazine hinge on its ability to inhibit persistent or late Na+ currents in a variety of voltage-gated sodium channels. Extracellular acidosis, typical of ischemic events, may alter the efficiency of drug/channel interactions. In this study, we examined pH modulation of ranolazines interaction with the cardiac sodium channel, Nav1.5. We performed whole-cell path clamp experiments at extracellular pH 7.4 and 6.0 on Nav1.5 transiently expressed in HEK293 cell line. Consistent with previous studies, we found that ranolazine induced a stable conformational state in the cardiac sodium channel with onset/recovery kinetics and voltage-dependence resembling intrinsic slow inactivation. This interaction diminished the availability of the channels in a voltage- and use-dependent manner. Low extracellular pH impaired inactivation states leading to an increase in late Na+ currents. Ranolazine interaction with the channel was also slowed 4–5 fold. However, ranolazine restored the voltage-dependent steady-state availability profile, thereby reducing window/persistent currents at pH 6.0 in a manner comparable to pH 7.4. These results suggest that ranolazine is effective at therapeutically relevant concentrations (10 μM), in acidic extracellular pH, where it compensates for impaired native slow inactivation.

Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants

The E1784K mixed syndrome mutant of the cardiac voltage‐gated sodium channel, NaV1.5, responds differently to temperature changes compared to the R1193Q mutant and wild‐type (WT) NaV1.5. In E1784K, elevated temperature causes a larger increase in persistent current; there is also an increase in use‐dependent inactivation at 1 Hz, which is not apparent at 3 Hz. WT NaV1.5 and R1193Q channels respond similarly to temperature changes. Action potential modelling (from extrapolated temperature coefficient (Q10)values) predicts the effects of differential temperature sensitivity on the cardiac action potential: greater attenuation of the epicardial action potential occurs in E1784K as temperature shifts from hypothermic to hyperthermic conditions, and when transient outward potassium currents are increased. The results from the action potential model predict that, at febrile temperatures, E1784K channels results in a larger transmural voltage gradient. Hyperthermia exacerbates the Brugada syndrome 1 (BrS1) phenotype, which may be arrhythmogenic in E1784K mutants.

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.

Temperature-dependent changes in neuronal dynamics in a patient with an SCN1A mutation and hyperthermia induced seizures

Dravet syndrome is the prototype of SCN1A-mutation associated epilepsies. It is characterised by prolonged seizures, typically provoked by fever. We describe the evaluation of an SCN1A mutation in a child with early-onset temperature-sensitive seizures. The patient carries a heterozygous missense variant (c3818C > T; pAla1273Val) in the NaV1.1 brain sodium channel. We compared the functional effects of the variant vs. wild type NaV1.1 using patch clamp recordings from channels expressed in Chinese Hamster Ovary Cells at different temperatures (32, 37, and 40 °C). The variant channels produced a temperature-dependent destabilization of activation and fast inactivation. Implementing these empirical abnormalities in a computational model predicts a higher threshold for depolarization block in the variant, particularly at 40 °C, suggesting a failure to autoregulate at high-input states. These results reveal direct effects of abnormalities in NaV1.1 biophysical properties on neuronal dynamics. They illustrate the value of combining cellular measurements with computational models to integrate different observational scales (gene/channel to patient).