Mohamed Chahine

Sodium Channel Mutations in Paramyotonia Congenita Uncouple Inactivation from Activation

Mutations in the adult human skeletal muscle Na+ channel alpha subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation. Increase of extracellular pH made the rate of inactivation of R1448H similar to that of R1448C, suggesting that this residue has an extracellular location and that its charge is important for normal inactivation. Analysis of single-channel data reveals that mutant channels inactivate normally from closed states, but poorly from the open state. The data suggest a critical role for the S4 helix of domain 4 in coupling between activation and inactivation.

New Insights into Cardiac and Brain Sodium Channels Modulation by Beta Blockers

Beta-adrenergic blocking agents known as beta blockers are widely used in clinical practice to treat several cardiovascular disorders such as hypertension and high blood pressure in general. They are also used as cardioprotective agents in post myocardial infarction, for the treatment of cardiac arrhythmias and were reported to be beneficial in treating migraine. Recently, they were shown to be efficacious in treating patients with several types of congenital long QT syndromes and in patient with catecholaminergic polymorphic ventricular tachycardia. They mainly act by antagonizing the effects of norepinephrine released from sympathetic nerve endings on beta adrenoceptors. A direct interaction with ion channels in addition to the beta blocking property is now becoming more and more accepted in the scientific community. Propranolol is the beta blocker prototype, it is commonly used as racemic mixture with equal concentrations of R-(+)- and S-(−)-enantiomers. Although, they have been classified as class II antiarrhythmic drugs by Vaughan Williams the molecular mechanisms by which they act is not fully elucidated. On the other hand, their beneficial effect in preventing migraine is not well understood. Earlier electrophysiological studies have reported the effects of propranolol on heart rate and conduction properties in frog auricular fibers, rat, and canine ventricular myocytes. Recent data have shown that beta blockers could modulate Nav1.5, the cardiac voltage-gated sodium channels, but the effect on the expressed brain sodium channels was not envisaged. In the paper by Wang et al., the authors studied the effect of propanol on heterologously expressed recombinant human cardiac (Nav1.5) and the three brain (Nav1.1, Nav1.2, and Nav1.3) sodium channels using whole-cell patch clamp recordings. Previous work from the authors group showed that racemic propranolol and R-(+)-propranolol block Nav1.5 channels (Wang et al., 2008). In this paper the authors extended their study to evaluate the molecular mechanism of the reported block. Both R-(+) and S-(−) propranolol block Nav1.5 sodium channels in tonic and phasic (use-dependent or frequency-dependent) manner with similar affinities. However, nadolol a non-selective beta blocker and metoprolol a selective beta 1 blocker did not induce any tonic or phasic block, suggesting that the sodium channel block property is not common to all beta blockers. More detailed biophysical studies from the authors revealed that that R-(+)-propranolol exhibits biophysical effects on Nav1.5 that are similar but not identical to lidocaine, the class 1 antiarrhythmic drug prototype. That R-(+)-propranolol acts as a typical local anesthetic and class 1 antiarrhythmics on sodium channels by interacting with specific residues in the DIV-S6 segment, including the phenylalanine-1760, known to play a central role in drug binding (Ragsdale et al., 1994) and therefore shares this property with established antiarrhythmic drugs. Finally, further detailed biophysical study from the authors showed that the brain sodium channels (Nav1.1, Nav1.2, and Nav1.3) exhibit less sensitivity to R-(+)-propranolol than the Nav1.5 channels. Since the phenylalanine-1760 is a conserved residue in all sodium channels, including brain sodium channels, studies to elucidate the basis of this reduced affinity at molecular level are warranted. These data reported by Wang et al., in this issue of Frontiers in Pharmacology of Ion Channel and Channelopathies, will pave the path toward a more understanding of the effect of beta blockers on sodium channels, a widely used class of drugs.

SCN5A Polymorphism Restores Trafficking of a Brugada Syndrome Mutation on a Separate Gene

Background— Brugada syndrome is associated with a high risk of sudden cardiac death and is caused by mutations in the cardiac voltage-gated sodium channel gene. Previously, the R282H-SCN5A mutation in the sodium channel gene was identified in patients with Brugada syndrome. In a family carrying the R282H-SCN5A mutation, an asymptomatic individual had a common H558R-SCN5A polymorphism and the mutation on separate chromosomes. Therefore, we hypothesized that the polymorphism could rescue the mutation. Methods and Results— In heterologous cells, expression of the mutation alone did not produce sodium current. However, coexpressing the mutation with the polymorphism produced significantly greater current than coexpressing the mutant with the wild-type gene, demonstrating that the polymorphism rescues the mutation. Using immunocytochemistry, we demonstrated that the R282H-SCN5A construct can traffic to the cell membrane only in the presence of the H558R-SCN5A polymorphism. Using fluorescence resonance energy transfer and protein fragments centered on H558R-SCN5A, we demonstrated that cardiac sodium channels preferentially interact when the polymorphism is expressed on one protein but not the other. Conclusions— This study suggests a mechanism whereby the Brugada syndrome has incomplete penetrance. More importantly, this study suggests that genetic polymorphisms may be a potential target for future therapies aimed at rescuing specific dysfunctional protein channels.

Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes

UNLABELLED Familial long QT syndrome (LQTS) and Brugada syndrome are two distinct human hereditary cardiac diseases known to cause ventricular tachyarrhythmias (torsade de pointes) and idiopathic ventricular fibrillation, respectively, which can both lead to sudden death. OBJECTIVE In this study we have identified and electrophysiologically characterized, in patients having either LQTS or Brugada syndrome, three mutations in SCN5A (a cardiac sodium channel gene). METHOD The mutant channels were expressed in a mammalian expression system and studied by means of the patch clamp technique. RESULTS The R1512W mutation found in our first patient diagnosed with Brugada syndrome produced a slowing of both inactivation and recovery from inactivation. The R4132G mutation found in our second patient who also presented Brugada syndrome, resulted in no measurable sodium currents. Both Brugada syndrome patients showed ST segment elevation and right bundle-branch block, and had experienced syncopes. The E1784K mutation found in the LQTS showed a persistent inward sodium current, a hyperpolarized shift of the steady-sate inactivation and a faster recovery from inactivation. CONCLUSION The different clinical manifestations of these three mutations most probably originate from the distinct electrophysiological abnormalities of the mutant cardiac sodium channels reported in this study.

A newly characterized SCN5A mutation underlying Brugada syndrome unmasked by hyperthermia

Febrile illness has been rarely reported to modulate ST segment elevation in right precordial leads on ECG or even precipitate ventricular fibrillation in patients with Brugada syndrome. We report the case of a patient whose Brugada ECG pattern was unmasked by hyperthermia secondary to acute cholangitis. Serial ECGs showed progressive attenuation of ST segment elevation as body temperature gradually returned to normal. Structural heart disease was ruled out. Intravenous flecainide injection reproduced a less remarkable ST segment elevation. Genetic screening demonstrated a single amino acid substitution (H681P) in the SCN5A gene, thus confirming the diagnosis of Brugada syndrome. In vitro expression of this newly characterized genetic defect revealed novel biophysical abnormalities consisting of a shift in both steady‐state activation and inactivation, resulting in a 60% reduction of sodium window current. Thus, SCN5A‐H681P mutation induces a significant loss of transmembrane current and is clinically associated with a pathologic phenotype that is elicited by hyperthermia. Overall the observed clinical features are in agreement with previous observations and strongly suggest that fever may be an environmental modifier among Brugada syndrome patients with a detrimental (and possibly arrhythmogenic) effect on cardiac repolarization. (J Cardiovasc Electrophysiol, Vol. 14, pp. 407‐411, April 2003)

Enzyme Domain Affects the Movement of the Voltage Sensor in Ascidian and Zebrafish Voltage-sensing Phosphatases

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN).The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

SCN5A mutation (T1620M) causing Brugada syndrome exhibits different phenotypes when expressed in Xenopus oocytes and mammalian cells

Brugada syndrome is a hereditary cardiac disease causing abnormal ST segment elevation in the ECG, right bundle branch block, ventricular fibrillation and sudden death. In this study we characterized a new mutation in the SCN5A gene (T1620M), causing the Brugada syndrome. The mutated channels were expressed in both Xenopus leavis oocytes and in mammalian tsA201 cells with and without the β‐subunit and studied using the patch clamp technique. Opposite phenotypes were observed depending on the expression system. T1620M mutation led to a faster recovery from inactivation and a shift of steady‐state inactivation to more positive voltages when expressed in Xenopus oocytes. However, using the mammalian expression system no effect on steady‐state inactivation was observed, but this mutation led to a slower recovery from inactivation. Our finding supports the idea that the slower recovery from inactivation of the cardiac sodium channels seen in our mammalian expression system could decrease the density of sodium channels during the cardiac cycle explaining the in vivo arrhythmogenesis in patients with Brugada syndrome.

Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes.

Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada syndrome has so far been associated with only one gene, SCN5A, which encodes the cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes, including the SCN5A, are implicated. The substitution (D1790G) causes LQTS and the insertion (D1795) induces both LQTS and Brugada syndromes in carrier patients. hH1/insD1795 and hH1/D1790G mutant channels were expressed in the tsA201 human cell line and characterized using the patch clamp technique in whole‐cell configuration. Our data revealed a persistent inward sodium current of about 6% at −30 mV for both D1790G and insD1795, and a reduction of 62% of channel expression for the insD1795. Moreover, a shift of steady‐state inactivation curve in both mutants was also observed. Our findings uphold the idea that LQT3 is related to a persistent sodium current whereas reduction in the expression level of cardiac sodium channels is one of the biophysical characteristics of Brugada syndrome.

A Proton Leak Current through the Cardiac Sodium Channel Is Linked to Mixed Arrhythmia and the Dilated Cardiomyopathy Phenotype

Cardiac Na+ channels encoded by the SCN5A gene are essential for initiating heart beats and maintaining a regular heart rhythm. Mutations in these channels have recently been associated with atrial fibrillation, ventricular arrhythmias, conduction disorders, and dilated cardiomyopathy (DCM). We investigated a young male patient with a mixed phenotype composed of documented conduction disorder, atrial flutter, and ventricular tachycardia associated with DCM. Further family screening revealed DCM in the patients mother and sister and in three of the mothers sisters. Because of the complex clinical phenotypes, we screened SCN5A and identified a novel mutation, R219H, which is located on a highly conserved region on the fourth helix of the voltage sensor domain of Nav1.5. Three family members with DCM carried the R219H mutation. The wild-type (WT) and mutant Na+ channels were expressed in a heterologous expression system, and intracellular pH (pHi) was measured using a pH-sensitive electrode. The biophysical characterization of the mutant channel revealed an unexpected selective proton leak with no effect on its biophysical properties. The H+ leak through the mutated Nav1.5 channel was not related to the Na+ permeation pathway but occurred through an alternative pore, most probably a proton wire on the voltage sensor domain. We propose that acidification of cardiac myocytes and/or downstream events may cause the DCM phenotype and other electrical problems in affected family members. The identification of this clinically significant H+ leak may lead to the development of more targeted treatments.

Voltage-gated sodium channels in neurological disorders.

Voltage-gated sodium channels play an essential biophysical role in many excitable cells such as neurons. They transmit electrical signals through action potential (AP) generation and propagation in the peripheral (PNS) and central nervous systems (CNS). Each sodium channel is formed by one alpha-subunit and one or more beta-subunits. There is growing evidence indicating that mutations, changes in expression, or inappropriate modulation of these channels can lead to electrical instability of the cell membrane and inappropriate spontaneous activity observed during pathological states. This review describes the biochemical, biophysical and pharmacological properties of neuronal voltage-gated sodium channels (VGSC) and their implication in several neurological disorders.