Pascal Gosselin-Badaroudine

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.

Gating pore currents and the resting state of Nav1.4 voltage sensor domains

Mammalian voltage-gated sodium channels are composed of four homologous voltage sensor domains (VSDs; DI, DII, DIII, and DIV) in which their S4 segments contain a variable number of positively charged residues. We used single histidine (H) substitutions of these charged residues in the Nav1.4 channel to probe the positions of the S4 segments at hyperpolarized potentials. The substitutions led to the formation of gating pores that were detected as proton leak currents through the VSDs. The leak currents indicated that the mutated residues are accessible from both sides of the membrane. Leak currents of different magnitudes appeared in the DI/R1H, DII/R1H, and DIII/R2H mutants, suggesting that the resting state position of S4 varies depending on the domain. Here, DI/R1H indicates the first arginine R1, in domain DI, has been mutated to histidine. The single R1H, R2H, and R3H mutations in DIV did not produce appreciable proton currents, indicating that the VSDs had different topologies. A structural model of the resting states of the four VSDs of Nav1.4 relaxed in their membrane/solution environment using molecular dynamics simulations is proposed based on the recent NavAb sodium channel X-ray structure. The model shows that the hydrophobic septa that isolate the intracellular and the extracellular media within the DI, DII, and DIII VSDs are ∼2 Å long, similar to those of Kv channels. However, the septum of DIV is longer, which prevents water molecules from hydrating the center of the VSD, thus breaking the proton conduction pathway. This structural model rationalizes the activation sequence of the different VSDs of the Nav1.4 channel.

Biophysics, pathophysiology, and pharmacology of ion channel gating pores

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.

Gating pore currents are defects in common with two Nav1.5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy.

Nav1.5 channels bearing voltage-sensor domain mutations associated with atypical cardiac arrhythmias and dilated cardiomyopathy generate gating pore currents.

Nav1.5 mutations linked to dilated cardiomyopathy phenotypes: Is the gating pore current the missing link?

Nav 1.5 dysfunctions are commonly linked to rhythms disturbances that include type 3 long QT syndrome (LQT3), Brugada syndrome (BrS), sick sinus syndrome (SSS) and conduction defects. Recently, this channel protein has been also linked to structural heart diseases such as dilated cardiomyopathy (DCM).Nav1.5 dysfunctions are commonly linked to rhythms disturbances that include type 3 long QT syndrome (LQT3), Brugada syndrome (BrS), sick sinus syndrome (SSS) and conduction defects. Recently, this channel protein has been also linked to structural heart diseases such as dilated cardiomyopathy (DCM).

Mutations in the Voltage Sensors of Domains I and II of Nav1.5 that are Associated with Arrhythmias and Dilated Cardiomyopathy Generate Gating Pore Currents

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.

Molecular biology and biophysical properties of ion channel gating pores

The voltage sensitive domain (VSD) is a pivotal structure of voltage-gated ion channels (VGICs) and plays an essential role in the generation of electrochemical signals by neurons, striated muscle cells, and endocrine cells. The VSD is not unique to VGICs. Recent studies have shown that a VSD regulates a phosphatase. Similarly, Hv1, a voltage-sensitive protein that lacks an apparent pore domain, is a self-contained voltage sensor that operates as an H⁺ channel. VSDs are formed by four transmembrane helices (S1-S4). The S4 helix is positively charged due to the presence of arginine and lysine residues. It is surrounded by two water crevices that extend into the membrane from both the extracellular and intracellular milieus. A hydrophobic septum disrupts communication between these water crevices thus preventing the permeation of ions. The septum is maintained by interactions between the charged residues of the S4 segment and the gating charge transfer center. Mutating the charged residue of the S4 segment allows the water crevices to communicate and generate gating pore or omega pore. Gating pore currents have been reported to underlie several neuronal and striated muscle channelopathies. Depending on which charged residue on the S4 segment is mutated, gating pores are permeant either at depolarized or hyperpolarized voltages. Gating pores are cation selective and seem to converge toward Eisenmanns first or second selectivity sequences. Most gating pores are blocked by guanidine derivatives as well as trivalent and quadrivalent cations. Gating pores can be used to study the movement of the voltage sensor and could serve as targets for novel small therapeutic molecules.

Sodium overload due to a persistent current that attenuates the arrhythmogenic potential of a novel LQT3 mutation

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 Ca2+ 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.

Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca2+ channel subfamily: CaV4

Insect DSC1 channels have sequences that are intermediate between voltage-gated Na+ and Ca2+ channels but have hitherto been classified as the former. Gosselin-Badaroudine et al. clone and characterize honeybee DSC1, revealing high selectivity for Ca2+ and suggesting reclassification of DSC1 homologues as Ca2+ channels.

Characterization of the honeybee AmNaV1 channel and tools to assess the toxicity of insecticides.

Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee’s sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee’s channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.