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Voltage gated sodium channels (Nav) are transmembrane proteins responsible for action potential initiation. Mutations mainly located in the voltage sensor domain (VSD) of Nav1.5, the cardiac sodium channel, have been associated with the development of arrhythmias combined with dilated cardiomyopathy. Gating pore currents have been observed with three unrelated mutations associated with similar clinical phenotypes. However, gating pores have never been associated with mutations outside the first domain of Nav1.5. The aim of this study was to explore the possibility that gating pore currents might be caused by the Nav1.5 R225P and R814W mutations (R3, S4 in DI and DII, respectively), which are associated with rhythm disturbances and dilated cardiomyopathy. Nav1.5 WT and mutant channels were transiently expressed in tsA201 cells. The biophysical properties of the alpha pore currents and the presence of gating pore currents were investigated using the patch-clamp technique. We confirmed the previously reported gain of function of the alpha pores of the mutant channels, which mainly consisted of increased window currents mostly caused by shifts in the voltage dependence of activation. We also observed gating pore currents associated with the R225P and R814W mutations. This novel permeation pathway was open under depolarized conditions and remained temporarily open at hyperpolarized potentials after depolarization periods. Gating pore currents could represent a molecular basis for the development of uncommon electrical abnormalities and changes in cardiac morphology. We propose that this biophysical defect be routinely evaluated in the case of Nav1.5 mutations on the VSD.

The transmembrane S6 segments of Na(+) sodium channels form the cytoplasmic entrance of the channel and line the internal aspects of the aqueous pore. This region of the channel has been implicated in Na(+) channel permeation, gating, and pharmacology. In this study we utilized cysteine substitutions and methanethiosulfonate reagent (MTSET) to investigate the role of the S6 segment of homologous domain 4 (D4S6) in the gating of the cardiac (Nav1.5) channel. D4S6 cysteine mutants were heterologously expressed in tsA201 cells and currents recorded using whole-cell patch clamp. Internal MTSET reduced the peak Na(+) currents, induced hyperpolarizing shifts in steady-state inactivation and slowed the recovery of mutant channels with cysteines inserted near the middle (F1760C, V1763C) and C-terminus (Y1767C) of the D4S6. These findings suggested a link between the MTSET inhibition and fast inactivation. This was confirmed by expressing the V1763C and Y1767C mutations in non-inactivating Nav1.5 channels. Removing inactivation abolished the MTSET inhibition of the V1763C and Y1767C mutants. The data indicate that the MTSET-induced reduction in current primarily results from slower recovery from inactivation that produces hyperpolarizing shifts in fast inactivation and decreases the steady-state availability of the channels. This contrasted with a cysteine inserted near the C-terminus of the D4S6 (I1770C) where MTSET increased the persistent Na(+) current at depolarized voltages consistent with impaired fast inactivation. Covalent modification of D4S6 cysteines with MTSET adduct appears to reduce the mobility of the D4S6 segment and stabilize the channels in the fast inactivated state. These findings indicate that residues located near the middle and C-terminus of the D4S6 play an important role in fast inactivation.

Voltage sensor domains (VSDs) are a feature of voltage gated ion channels (VGICs) and voltage sensitive proteins. They are composed of four transmembrane (TM) segments (S1-S4). Currents leaking through VSDs are called omega or gating pore currents. Gating pores are caused by mutations of the highly conserved positively charged amino acids in the S4 segment that disrupt interactions between the S4 segment and the gating charge transfer center (GCTC). The GCTC separates the intracellular and extracellular water crevices. The disruption of S4-GCTC interactions allows these crevices to communicate and create a fast activating and non-inactivating alternative cation-selective permeation pathway of low conductance, or a gating pore. Gating pore currents have recently been shown to cause periodic paralysis phenotypes. There is also increasing evidence that gating pores are linked to several other familial diseases. For example, gating pores in Nav1.5 and Kv7.2 channels may underlie mixed arrhythmias associated with dilated cardiomyopathy (DCM) phenotypes and peripheral nerve hyperexcitability (PNH), respectively. There is little evidence for the existence of gating pore blockers. Moreover, it is known that a number of toxins bind to the VSD of a specific domain of Na(+) channels. These toxins may thus modulate gating pore currents. This focus on the VSD motif opens up a new area of research centered on developing molecules to treat a number of cell excitability disorders such as epilepsy, cardiac arrhythmias, and pain. The purpose of the present review is to summarize existing knowledge of the pathophysiology, biophysics, and pharmacology of gating pore currents and to serve as a guide for future studies aimed at improving our understanding of gating pores and their pathophysiological roles.

Long QT syndrome (LQTS) is a congenital abnormality of cardiac repolarization that manifests as a prolonged QT interval on 12-lead electrocardiograms (ECGs). The syndrome may lead to syncope and sudden death from ventricular tachyarrhythmias known as torsades de pointes. An increased persistent Na(+) current is known to cause a Ca(2+) overload in case of ischemia for example. Such increased Na(+) persistent current is also usually associated to the LQT3 syndrome. The purpose of this study was to investigate the pathological consequences of a novel mutation in a family affected by LQTS. The impact of biophysical defects on cellular homeostasis are also investigated. Genomic DNA was extracted from blood samples, and a combination of PCR and DNA sequencing of several LQTS-linked genes was used to identify mutations. The mutation was reproduced in vitro and was characterized using the patch clamp technique and in silico quantitative analysis. A novel mutation (Q1476R) was identified on the SCN5A gene encoding the cardiac Na(+) channel. Cells expressing the Q1476R mutation exhibited biophysical alterations, including a shift of SS inactivation and a significant increase in the persistent Na(+) current. The in silico analysis confirmed the arrhythmogenic character of the Q1476R mutation. It further revealed that the increase in persistent Na(+) current causes a frequency-dependent Na(+) overload in cardiomyocytes co-expressing WT and mutant Nav1.5 channels that, in turn, exerts a moderating effect on the lengthening of the action potential (AP) duration caused by the mutation. The Q1476R mutation in SCN5A results in a three-fold increase in the window current and a persistent inward Na(+) current. These biophysical defects may expose the carrier of the mutation to arrhythmias that occur preferentially in the patient at rest or during tachycardia. However, the Na(+) overload counterbalances the gain-of-function of the mutation and is beneficial in that it prevents severe arrhythmias at intermediate heart rates.

Mortality rate is high in psychiatric patients versus general population. An important cause of this increased mortality is sudden cardiac death (SCD) as a major side-effect of psychotropic drugs. These SCDs generally result from arrhythmias occurring when the posology is high and may attain a toxic threshold but also at dosages within therapeutic range, in the presence of risk factors. There are three kinds of risk factors: physiological (e.g., low cardiac rate of sportsmen), physiopathological (e.g., hepatic insufficiency, hypothyroidism) and "therapeutic" (due to interactions between psychotropic drugs and other medicines). Association of pharmacological agents may increase the likelihood of SCDs either by (i) a pharmacokinetic mechanism (e.g., increased torsadogenic potential of a psychotropic drug when its destruction and/or elimination are compromised) or (ii) a pharmacodynamical mechanism (e.g., mutual potentiation of proarrhythmic properties of two drugs). In addition, some psychotropic drugs may induce sudden death in cases of pre-existing congenital cardiopathies such as (i) congenital long QT syndrome, predisposing to torsade de pointes that eventually cause syncope and sudden death. (ii) A Brugada syndrome, that may directly cause ventricular fibrillation due to reduced sodium current through Nav1.5 channels. Moreover, psychotropic drugs may be a direct cause of cardiac lesions also leading to SCD. This is the case, for example, of phenothiazines responsible for ischemic coronaropathies and of clozapine that is involved in the occurrence of myocarditis. The aims of this work are to delineate: (i) the risk of SCD related to the use of psychotropic drugs; (ii) mechanisms involved in the occurrence of such SCD; (iii) preventive actions of psychotropic drugs side effects, on the basis of the knowledge of patient-specific risk factors, documented from clinical history, ionic balance, and ECG investigation by the psychiatrist.

The human cardiac sodium channel Na(v)1.5 encoded by the SCN5A gene plays a critical role in cardiac excitability and the propagation of action potentials. Na(v)1.5 dysfunctions due to mutations cause cardiac diseases such as the LQT3 form of long QT syndrome, conduction disorders, and Brugada syndrome (BrS). They have also recently been associated with dilated cardiomyopathy. BrS is characterized by coved ST-segment elevation on surface ECGs and lethal ventricular arrhythmias in an apparently structurally normal heart. Na(v)1.5 mutations that cause BrS result in a loss of channel function. Our aim was to functionally characterize two novel Na(v)1.5 mutations (A124D and V1378M) in BrS patients. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in tsA201 cells in the presence of the β(1)-auxiliary subunit. The patch-clamp technique and immunocytochemistry approaches were used to study the mutant channels and their cellular localization. The two mutant channels displayed a dramatic reduction in current density but had normal gating properties. The reduction in current density was caused by the retention of channel proteins in the endoplasmic reticulum (ER). Mutant channel retention could be partially reversed by incubating transfected cells at 25°C and by treating them with mexiletine (for V1378M but not A124D), or with curcumin or thapsigargin, two drugs that target ER resident proteins. It is likely that the clinical phenotypes observed in these two BrS patients were related to a surface expression defect caused by ER retention.

Voltage-gated sodium Na(+) channels are membrane-bound proteins incorporating aqueous conduction pores that are highly selective for sodium Na(+) ions. The opening of these channels results in the rapid influx of Na(+) ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na(+)-selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary β subunits (β(1), β(2)) were first described. Subsequent cloning and heterologous expression studies revealed that the α subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, 10 isoforms of the Na(+) channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four β subunits (β(1)-β(4)) and two splice variants (β(1A), β(1B)) have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of β subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.