Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5

Chatin, Benoît; Colombier, Pauline; Gamblin, Anne Laure; Allouis, Marie; Bouffant, Françoise Le

doi:10.1042/bj20140604

Cited by 9 publications

(7 citation statements)

References 48 publications

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“…Cotransfection of p50 increased the I Kir2.1 at potentials between −120 and −90 mV and between −70 and −50 mV (Figures 5B,D ; P < 0.05), suggesting that the dynein/dynactin complex is involved in the retrograde trafficking of Kir2.1 channels. Conversely, p50 reduced the I Nav1.5 at potentials ranging −40 and +30 mV in cells expressing Nav1.5 channels alone (Figures 5C,E ; P < 0.05), thus confirming previous results that demonstrate the role of the dynein/dynactin complex in the anterograde trafficking of Nav1.5 channels (Chatin et al, 2014 ). When Kir2.1 and Nav1.5 channels were expressed together, both I Kir2.1 and I Nav1.5 were significantly increased as expected (Figures 5B–E ).…”

Section: Resultssupporting

confidence: 90%

“…The experiments conducted in cells overexpressing the p50 subunit to inhibit the dynein/dynamitin motor complex showed that it is involved in the retrograde and anterograde trafficking of Kir2.1 and Nav1.5 channels, respectively. Our results confirm previous data showing that p50 interacts with Nav1.5 channels reducing their expression at the cell surface and the I Nav1.5 density (Chatin et al, 2014 ). Importantly, our results also suggested that the dynein/dynamitin motor complex is critical for the anterograde trafficking of the pool of Kir2.1 and Nav1.5 channels that form the complexes, supporting the hypothesis that their biology may be similar to that of Nav1.5 channels (Figure 9 ).…”

Section: Discussionsupporting

confidence: 93%

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Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone

et al. 2017

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Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

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Section: Resultssupporting

confidence: 90%

Section: Discussionsupporting

confidence: 93%

Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone

et al. 2017

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“…Furthermore, it has been demonstrated that activation of SAR1 leads to the recruitment and internalization of Na v 1.5 cargo into the coated transition vesicle COPII-coated vesicles that will ensure its ER-to-Golgi trafficking [202]. The Na v 1.5 ER export is also controlled by Dynamitin as demonstrated by Chatin et al, who have proved, using a yeast two-hybrid system, that Dynamitin (C-terminal domain), interacted with the Na v 1.5 DI-DII linker between amino acids 417 and 444 and that this interaction is crucial for the Na v 1.5 cell-surface density probably through controlling the ER-to-Golgi trafficking [203].…”

Section: Regulation Of the Er-to-golgi Traffickingmentioning

confidence: 85%

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias

Daimi

Lozano-Velasco

Aránega

et al. 2022

IJMS

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Nav1.5 is the predominant cardiac sodium channel subtype, encoded by the SCN5A gene, which is involved in the initiation and conduction of action potentials throughout the heart. Along its biosynthesis process, Nav1.5 undergoes strict genomic and non-genomic regulatory and quality control steps that allow only newly synthesized channels to reach their final membrane destination and carry out their electrophysiological role. These regulatory pathways are ensured by distinct interacting proteins that accompany the nascent Nav1.5 protein along with different subcellular organelles. Defects on a large number of these pathways have a tremendous impact on Nav1.5 functionality and are thus intimately linked to cardiac arrhythmias. In the present review, we provide current state-of-the-art information on the molecular events that regulate SCN5A/Nav1.5 and the cardiac channelopathies associated with defects in these pathways.

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“…To this end, the yeasts transformed with bait and prey vectors were plated onto a single selective medium lacking Leu-Trp (SC-Leu-Trp) plate at 30°C for 18 h. Afterwards, replicas were sequentially plated (a first incubation at 30°C for 24 h followed by a second incubation at 30°C for 1 d) onto selection plates in the presence of SC-Leu-Trp-His-3AT or SC-Leu-Trp-5FOA. Densitometric analysis of the images was performed using ImageJ to analyze putative changes in the SUR2A–AnkB interaction induced by the p.S1054Y mutation ( Chatin et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

A Cantú syndrome mutation produces dual effects on KATP channels by disrupting ankyrin B regulation

Crespo-García

Rubio-Alarcón

Cámara-Checa

et al. 2022

Journal of General Physiology

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ATP-sensitive potassium (KATP) channels composed of Kir6.x and sulfonylurea receptor (SURs) subunits couple cellular metabolism to electrical activity. Cantú syndrome (CS) is a rare disease caused by mutations in the genes encoding Kir6.1 (KCNJ8) and SUR2A (ABCC9) that produce KATP channel hyperactivity due to a reduced channel block by physiological ATP concentrations. We functionally characterized the p.S1054Y SUR2A mutation identified in two CS carriers, who exhibited a mild phenotype although the mutation was predicted as highly pathogenic. We recorded macroscopic and single-channel currents in CHO and HEK-293 cells and measured the membrane expression of the channel subunits by biotinylation assays in HEK-293 cells. The mutation increased basal whole-cell current density and at the single-channel level, it augmented opening frequency, slope conductance, and open probability (Po), and promoted the appearance of multiple conductance levels. p.S1054Y also reduced Kir6.2 and SUR2A expression specifically at the membrane. Overexpression of ankyrin B (AnkB) prevented these gain- and loss-of-function effects, as well as the p.S1054Y-induced reduction of ATP inhibition of currents measured in inside-out macropatches. Yeast two-hybrid assays suggested that SUR2A WT and AnkB interact, while p.S1054Y interaction with AnkB is decreased. The p.E322K Kir6.2 mutation, which prevents AnkB binding to Kir6.2, produced similar biophysical alterations than p.S1054Y. Our results are the first demonstration of a CS mutation whose functional consequences involve the disruption of AnkB effects on KATP channels providing a novel mechanism by which CS mutations can reduce ATP block. Furthermore, they may help explain the mild phenotype associated with this mutation.

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Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5

Cited by 9 publications

References 48 publications

Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone

Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias

A Cantú syndrome mutation produces dual effects on KATP channels by disrupting ankyrin B regulation

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