scholarly journals Calmodulin limits pathogenic Na+ channel persistent current

2017 ◽  
Vol 149 (2) ◽  
pp. 277-293 ◽  
Author(s):  
Haidun Yan ◽  
Chaojian Wang ◽  
Steven O. Marx ◽  
Geoffrey S. Pitt
Keyword(s):  
Cardiac Arrhythmias ◽  
Persistent Current ◽  
Purkinje Neurons ◽  
Na Channel ◽  
Wild Type ◽  
Na Current ◽  
Voltage Gated ◽  
Nav Channels ◽  
Cerebellar Purkinje Neurons ◽  
Terminal Domains

Increased “persistent” current, caused by delayed inactivation, through voltage-gated Na+ (NaV) channels leads to cardiac arrhythmias or epilepsy. The underlying molecular contributors to these inactivation defects are poorly understood. Here, we show that calmodulin (CaM) binding to multiple sites within NaV channel intracellular C-terminal domains (CTDs) limits persistent Na+ current and accelerates inactivation across the NaV family. Arrhythmia or epilepsy mutations located in NaV1.5 or NaV1.2 channel CTDs, respectively, reduce CaM binding either directly or by interfering with CTD–CTD interchannel interactions. Boosting the availability of CaM, thus shifting its binding equilibrium, restores wild-type (WT)–like inactivation in mutant NaV1.5 and NaV1.2 channels and likewise diminishes the comparatively large persistent Na+ current through WT NaV1.6, whose CTD displays relatively low CaM affinity. In cerebellar Purkinje neurons, in which NaV1.6 promotes a large physiological persistent Na+ current, increased CaM diminishes the persistent Na+ current, suggesting that the endogenous, comparatively weak affinity of NaV1.6 for apoCaM is important for physiological persistent current.

scholarly journals Intrinsic Mechanisms in the Gating of Resurgent Na+ Currents

2021 ◽  
Author(s):  
Joseph L. Ransdell ◽  
Jonathan D. Moreno ◽  
Druv Bhagavan ◽  
Jonathan R. Silva ◽  
Jeanne M. Nerbonne
Keyword(s):  
Voltage Clamp ◽  
Sodium Current ◽  
Neuronal Cell ◽  
Purkinje Neurons ◽  
Relative Distribution ◽  
Membrane Hyperpolarization ◽  
Voltage Gated ◽  
Nav Channels ◽  
Cerebellar Purkinje Neurons ◽  
Kinetics Of

ABSTRACTThe resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navβ4 channel subunit, can modulate the amplitude of INaR.SUMMARYThe resurgent component of the voltage-gated sodium current (INaR) is revealed on membrane hyperpolarizations following brief depolarizing voltage steps that activate the rapidly activating and inactivating, transient Nav current (INaT). To probe the mechanisms contributing to the generation and properties of INaR, we combined whole-cell voltage-clamp recordings from mouse cerebellar Purkinje neurons with computational modeling to develop a novel, blocking particle-independent, model for the gating of INaR that involves two parallel inactivation pathways, and we show that this model recapitulates the detailed biophysical properties of INaR measured in mouse cerebellar Purkinje neurons.

A novel mutation in transmembrane protein 168 causes fatal ventricular arrhythmogenesis in Brugada syndrome

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Ogita ◽  
D.P Zankov ◽  
A Shimizu
Keyword(s):  
Ventricular Tachycardia ◽  
Current Density ◽  
Brugada Syndrome ◽  
Transmembrane Protein ◽  
Left Ventricular ◽  
Public Institution ◽  
Na Channel ◽  
Funding Source ◽  
Wild Type ◽  
Na Current

Abstract   Brugada syndrome (BrS) is diagnosed by a typical electrocardiography (ECG) with ST-segment elevation in precordial leads and tends to induce sudden cardiac death (SCD) due to ventricular tachycardia/fibrillation. About 20% of SCDs in non-structural cardiac diseases are considered to be caused by BrS. In patients with BrS, loss of function mutations in the Na+ channel is often observed, but the causative gene mutation is not detected for about 70% of BrS patients. Here, we investigated a family with clinically diagnosed BrS, in which no known gene mutations related to BrS had not been found, by whole exome sequencing. Novel heterozygous variant (c. 1616G>A, p. R539Q) in transmembrane protein 168 (TMEM168) was identified only in symptomatic family members. Similar to endogenous TMEM168, both wild-type and mutant TMEM168 localized at the nuclear membrane. Na+ current density in whole-cell patch-clamp recordings was significantly reduced in HL-1 cardiomyocytes transfected with TMEM168 R539Q mutant, compared with those with wild-type TMEM168. Next, heterozygous Tmem168 1616G>A knock-in mice were generated by the CRISPR/Cas9 genome editing technology. Although the knock-in mice had no abnormalities in ECG at the physiological state, the treatment with ajmaline caused various arrhythmias including ventricular tachycardia/fibrillation in the knock-in mice, but not in wild-type mice. Na+ current density and the parameters of action potentials were remarkably impaired in the cardiomyocytes of the knock-in mice. Optical mapping analysis in the whole heart showed the reduced left ventricular conduction velocity in the knock-in mice. The expression of Nav1.5, an α-subunit of the cardiac Na+ channel, was significantly decreased in the mutant TMEM168-transfected HL-1 cells and the knock-in hearts. We found that the decrease was caused by the enhanced ubiquitination of Nav1.5, which was mediated by increased binding of Nedd4–2 E3 ubiquitin ligase to Nav1.5 in the knock-in hearts. Co-immunoprecipitation experiments demonstrated that overactivity of Nedd4–2 is a result of Tmem168 mutant-mediated sequestration of a chaperon protein αB-crystallin, a Nav1.5-binding molecule that interferes with the interaction of Nedd4–2 with Nav1.5. These findings reveal the molecular mechanism of TMEM168 R539Q mutation-induced fatal ventricular arrhythmias in BrS. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): JSPS Grants-in-aid for Scientific Research

scholarly journals Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Paul G DeCaen ◽  
Yuka Takahashi ◽  
Terry A Krulwich ◽  
Masahiro Ito ◽  
David E Clapham
Keyword(s):  
Divalent Cations ◽  
Ion Selectivity ◽  
Resting Membrane Potential ◽  
Na Channel ◽  
Ph Homeostasis ◽  
Ionic Selectivity ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Alkaliphilic Bacillus

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na+ to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na+ channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na+, Ca2+ and K+ ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na+ or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

scholarly journals Abnormal Na channel gating in murine cardiac myocytes deficient in myotonic dystrophy protein kinase

2003 ◽  
Vol 12 (2) ◽  
pp. 147-157 ◽  
Author(s):  
Hwa C. Lee ◽  
Manoj K. Patel ◽  
Dilawaar J. Mistry ◽  
Qingcai Wang ◽  
Sita Reddy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Current Density ◽  
Channel Gating ◽  
Resting Membrane Potential ◽  
Na Channel ◽  
Na Channels ◽  
Wild Type ◽  
Membrane Excitability ◽  
Na Current ◽  
Deficient Mice

DMPK is a serine/threonine kinase implicated in the human disease myotonic muscular dystrophy (DM). Skeletal muscle Na channels exhibit late reopenings in Dmpk-deficient mice and peak current density is reduced, implicating DMPK in regulation of membrane excitability. Since complete heart block and sudden cardiac death occur in the disease, we tested the hypothesis that cardiac Na channels also exhibit abnormal gating in Dmpk-deficient mice. We made whole cell and cell-attached patch clamp recordings of ventricular cardiomyocytes enzymatically isolated from wild-type, Dmpk+/−, and Dmpk−/− mice. Recordings from membrane patches containing one or a few Na channels revealed multiple Na channel reopenings occurring after the macroscopic Na current had subsided in both Dmpk+/− and Dmpk−/− muscle, but only rare reopenings in wild-type muscle (>3-fold difference, P < 0.05). This resulted in a plateau of non-inactivating Na current in Dmpk-deficient muscle. The magnitude of this plateau current was independent on the magnitude of the test potential from −40 to 0 mV and was also independent of gene dose. Macroscopic Na current density was similar in wild-type and Dmpk-deficient muscle, as was steady-state Na channel gating. Decay of macroscopic currents was slowed in Dmpk−/− muscle, but not in Dmpk+/− or wild-type muscle. Entry into, and recovery from, inactivation were similar at multiple test potentials in wild-type and Dmpk-deficient muscle. Resting membrane potential was depolarized, and action potential duration was significantly prolonged in Dmpk-deficient muscle. Thus in cardiac muscle, Dmpk deficiency results in multiple late reopenings of Na channels similar to those seen in Dmpk-deficient skeletal muscle. This is reflected in a plateau of non-inactivating macroscopic Na current and prolongation of cardiac action potentials.

scholarly journals Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

2013 ◽  
Vol 305 (4) ◽  
pp. H431-H445 ◽  
Author(s):  
Anthony W. Herren ◽  
Donald M. Bers ◽  
Eleonora Grandi
Keyword(s):  
Heart Failure ◽  
Na Channel ◽  
Macromolecular Complex ◽  
Na Current ◽  
Gene Encoding ◽  
Α Subunit ◽  
Post Translational Modifications ◽  
Voltage Gated ◽  
Novel Therapeutic Strategies ◽  
And Function

The voltage-gated Na channel isoform 1.5 (NaV1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding NaV1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the NaV1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that NaV1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered NaV1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies.

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

2016 ◽  
Vol 148 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Pascal Gosselin-Badaroudine ◽  
Adrien Moreau ◽  
Louis Simard ◽  
Thierry Cens ◽  
Matthieu Rousset ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Expression System ◽  
Selectivity Filter ◽  
Inactivation Kinetics ◽  
Na Channel ◽  
Biophysical Characterization ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Nav Channels

Bilaterian voltage-gated Na+ channels (NaV) evolved from voltage-gated Ca2+ channels (CaV). The Drosophila melanogaster Na+ channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between CaV and NaV channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca2+-permeable Na+ channel belonging to the NaV2 family because of its sequence similarity with NaV channels. This is despite insect NaV2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca2+ than Na+. In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd2+ and Zn2+. These characteristics are reminiscent of CaV channels. We also show a strong selectivity for Ca2+ and Ba2+ ions, marginal permeability to Li+, and impermeability to Mg2+ and Na+ ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as CaV4 rather than NaV2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee’s channel, namely slow activation and inactivation kinetics and strong selectivity for Ca2+ ions.

scholarly journals Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

2012 ◽  
Vol 140 (4) ◽  
pp. 435-454 ◽  
Author(s):  
Chien-Jung Huang ◽  
Laurent Schild ◽  
Edward G. Moczydlowski
Keyword(s):  
Na Channel ◽  
Ionic Selectivity ◽  
Voltage Gated ◽  
Trapped Ion ◽  
Toxin Binding ◽  
Outer Vestibule ◽  
Nav Channels ◽  
Na Ions ◽  
Gene Isoforms ◽  
Conformational Coupling

Voltage-gated Na+ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca2+ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca2+ or Na+ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca2+ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

scholarly journals Gjd2b-mediated gap junctions promote glutamatergic synapse formation and dendritic elaboration in Purkinje neurons

2020 ◽  
Author(s):  
Sahana Sitaraman ◽  
Gnaneshwar Yadav ◽  
Shaista Jabeen ◽  
Vandana Agarwal ◽  
Vatsala Thirumalai
Keyword(s):  
Gap Junctions ◽  
Dendritic Growth ◽  
Synapse Formation ◽  
Purkinje Neurons ◽  
Dendritic Arbor ◽  
Glutamatergic Synapse ◽  
Wild Type ◽  
Synapse Density ◽  
Chemical Synapses ◽  
Cerebellar Purkinje Neurons

AbstractGap junctions between neurons serve as electrical synapses, in addition to conducting metabolites and signaling molecules. These functions of gap junctions have led to the idea that during development, gap junctions could prefigure chemical synapses. We present evidence for this idea at a central, glutamatergic synapse and provide some mechanistic insights. Here, we show that reduction or loss of Gjd2b-containing gap junctions led to a decrease in glutamatergic synapse density in cerebellar Purkinje neurons (PNs) in larval zebrafish. Gjd2b-/- larvae exhibited faster mEPSCs and a consistent decrease in dendritic arbor size. These PNs also showed decreased branch elongations but normal rate of branch retractions. Further, the dendritic growth deficits in gjd2b-/- mutants were rescued by expressing full length Gjd2b in single PNs. This suggests that Gjd2b may form heterotypic channels with other connexins in gjd2b-/- larvae, though it is not clear if PNs in wild type animals make homotypic or heterotypic gap junction channels. Dendritic growth deficits were not rescued by expressing a deletion mutant of Gjd2b unable to form functional channels. Finally, the expression levels of five isoforms of camkii were increased in gjd2b-/- larvae and inhibition of CaMKII restored dendritic arbor lengths of mutant larvae to wild type levels. These results suggest a link between signaling via Gjd2b-containing gap junctions, CaMKII function and dendritic growth. In sum, our results demonstrate that Gjd2b-mediated gap junctions are key regulators of glutamatergic synapse formation and dendritic elaboration in PNs.

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