Aurélien Chatelier

Functional characterization of a Ca2+‐activated non‐selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (Iti). Iti is the result of three different [Ca]i‐sensitive currents: the Na+–Ca2+ exchange current, a Ca2+‐activated chloride current and a Ca2+‐activated non‐selective cationic current. Using the cell‐free configuration of the patch‐clamp technique, we have characterized the properties of a Ca2+‐activated non‐selective cation channel (NSCCa) in freshly dissociated human atrial cardiomyocytes. In excised inside‐out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na+ and K+) and was slightly permeable to Ca2+ ions. The channels open probability was increased by depolarization and a rise in internal calcium, for which the Kd for [Ca2+]i was 20.8 μm. Channel activity was reduced in the presence of 0.5 mm ATP or 10 μm glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 mm flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the ‘TRP melastatin’ subfamily. In conclusion, the NSCCa channel is a serious candidate to support the delayed after‐depolarizations observed in [Ca2+] overload and thus may be implicated in the genesis of arrhythmias.

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.

A distinct de novo expression of Nav1.5 sodium channels in human atrial fibroblasts differentiated into myofibroblasts.

Fibroblasts play a major role in heart physiology. In pathological conditions, they can lead to cardiac fibrosis when they differentiate into myofibroblasts. This differentiated status is associated with changes in expression profile leading to neo‐expression of proteins such as ionic channels. The present study investigates electrophysiological changes associated with fibroblast differentiation focusing on voltage‐gated sodium channels in human atrial fibroblasts and myofibroblasts. We show that human atrial fibroblast differentiation in myofibroblasts is associated with de novo expression of voltage gated sodium current. Multiple arguments support that this current is predominantly supported by the Nav1.5 α‐subunit which may generate a persistent sodium entry into myofibroblasts. Our data revealed that Nav1.5 α‐subunit expression is not restricted to cardiac myocytes within the atrium. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this channel could influence myofibroblasts function.

Activation of the Ca2+-Activated Nonselective Cation Channel by Diacylglycerol Analogues in Rat Cardiomyocytes

Introduction: Cardiac hypertrophy is associated with changes in electrophysiologic properties due to ionic channel modifications and increases in protein kinase C (PKC) activity and diacylglycerol (DAG) content. These changes may contribute to an increased propensity for arrhythmia. Similar electrophysiologic modifications have been reported in adult rat cardiomyocytes undergoing dedifferentiation in primary culture.

The β1-Subunit of NaV1.5 Cardiac Sodium Channel is required for a Dominant Negative Effect through α-α Interaction

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Nav1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Nav1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Nav1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β1-subunit was essential for this dominant negative effect. Indeed, the absence of the β1-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β1-subunit was present. Our findings reveal a new role for β1-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.

Biophysical Properties of Human Nav1.7 Splice Variants and Their Regulation by Protein Kinase A

The sodium channel Na(v)1.7 is preferentially expressed in nociceptive neurons and is believed to play a crucial role in pain sensation. Four alternative splice variants are expressed in human dorsal root ganglion neurons, two of which differ in exon 5 by two amino acids in the S3 segment of domain I (exons 5A and 5N). Two others differ in exon 11 by the presence (11L) or absence (11S) of an 11 amino acid sequence in the loop between domains I and II, an important region for PKA regulation. In the present study, we used the whole cell configuration of the patch-clamp technique to investigate the biophysical properties and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) modulation of these splice variants expressed in tsA201 cells in the presence of the beta(1)-subunit. The alternative splicing of Na(v)1.7 had no effect on most of the biophysical properties of this channel, including activation, inactivation, and recovery from inactivation. However, development of inactivation experiments revealed that the isoform containing exon 5A had slower kinetics of inactivation for negative potentials than that of the variant containing exon 5N. This difference was associated with higher ramp current amplitudes for isoforms containing exon 5A. Moreover, 8Br-cAMP-mediated phosphorylation induced a negative shift of the activation curve of variants containing exon 11S, whereas inactivation properties were unchanged. Isoforms with exon 11L were not modulated by 8Br-cAMP-induced phosphorylation. We conclude that alternative splicing of human Na(v)1.7 can specifically modulate the biophysical properties and cAMP-mediated regulation of this channel. Changing the proportions of these variants may thus influence neuronal excitability and pain sensation.

Biophysical characterisation of the persistent sodium current of the Nav1.6 neuronal sodium channel: a single-channel analysis

Nav1.6 is the major voltage-gated sodium channel at nodes of Ranvier. This channel has been shown to produce a robust persistent inward current in whole-cell experiments. Nav1.6 plays an important role in axonal conduction and may significantly contribute to the pathophysiology of the injured nervous system through this persistent current. However, the underlying molecular mechanisms and regulation of the persistent current are not well understood. Using the whole-cell configuration of the patch-clamp technique, we investigated the Nav1.6 transient and persistent currents in HEK-293. Previous studies have shown that the persistent current depended on the content of the patch electrode. Therefore, we characterised the single-channel properties of the persistent current with an intact intracellular medium using the cell-attached configuration of the patch-clamp technique. In HEK-293 cells, the Nav1.6 persistent current recorded in the whole-cell configuration was 3–5% of the peak transient current. In single-channel recording, the ratio between peak and persistent open probability confirmed the magnitude of the persistent current observed in the whole-cell configuration. The cell-attached configuration revealed that the molecular mechanism of the whole-cell persistent current is a consequence of single Nav1.6 channels reopening.

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states.

BACKGROUND Like many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors. METHODS Patch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation. RESULTS The present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current. CONCLUSIONS Our findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route. GENERAL SIGNIFICANCE This work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.

ANO1 contributes to Angiotensin-II-activated Ca2+-dependent Cl− current in human atrial fibroblasts

Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway.

Unmasked Brugada Pattern by Ajmaline Challenge in Patients with Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) generates missplicing of the SCN5A gene, encoding the cardiac sodium channel (Nav1.5). Brugada syndrome, which partly results from Nav1.5 dysfunction and causes increased VF occurrence, can be unmasked by ajmaline. We aimed to investigate the response to ajmaline challenge in DM1 patients and its potential impact on their sudden cardiac death risk stratification.