Sodium channel β1 subunits are post-translationally modified by tyrosine phosphorylation, S-palmitoylation, and regulated intramembrane proteolysis

Voltage-gated sodium channel (VGSC) β1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell–cell and cell–matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC β1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that β1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that β1 subunits undergo regulated intramembrane proteolysis via the activity of β-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, β1-ICD, which modulates transcription. Here, we report that β1 subunits are phosphorylated by FYN kinase. Moreover, we show that β1 subunits are S-palmitoylated. Substitution of a single residue in β1, Cys-162, to alanine prevented palmitoylation, reduced the level of β1 polypeptides at the plasma membrane, and reduced the extent of β1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of β1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of β1-p.C162A to WT levels. Despite these observations, palmitoylation-null β1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC β subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

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Membrane proteases and tetraspanins

Biochemical Society Transactions ◽

10.1042/bst0390541 ◽

2011 ◽

Vol 39 (2) ◽

pp. 541-546 ◽

Cited By ~ 13

Author(s):

María Yáñez-Mó ◽

Francisco Sánchez-Madrid ◽

Carlos Cabañas

Keyword(s):

Plasma Membrane ◽

Cross Talk ◽

Signalling Pathways ◽

Local Concentration ◽

Matrix Degradation ◽

Adhesion Receptors ◽

Intramembrane Proteolysis ◽

Regulated Intramembrane Proteolysis ◽

Protein Shedding ◽

Internalization Rate

TEMs (tetraspanin-enriched microdomains) are specialized platforms in the plasma membrane that include adhesion receptors and enzymes. Insertion into TEMs dictates the local concentration of these molecules, regulates their internalization rate, their interaction and cross-talk with other receptors at the plasma membrane and provides links with certain signalling pathways. We focus on the associations described for tetraspanins with membrane proteases and their substrates, reviewing the emerging evidence in the literature that suggests that TEMs might be essential platforms for regulating protein shedding, RIP (regulated intramembrane proteolysis) and matrix degradation and assembly.

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Astrocyte Ca2+ Signaling is Facilitated in an Scn1a+/− Mouse Model of Dravet Syndrome

10.1101/2021.05.18.444602 ◽

2021 ◽

Author(s):

Kouya Uchino ◽

Wakana Ikezawa ◽

Yasuyoshi Tanaka ◽

Masanobu Deshimaru ◽

Kaori Kubota ◽

...

Keyword(s):

Mouse Model ◽

Sodium Channel ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

Heterozygous Mutation ◽

Loss Of Function ◽

Cell Type ◽

Voltage Gated ◽

A Subunit ◽

The Brain

Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A, which encodes a subunit of the voltage-gated sodium channel, Nav1.1, in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a. We found that the slope of spontaneous Ca2+ spiking was increased without a change in amplitude in Scn1a+/− astrocytes. In addition, ATP-induced transient Ca2+ influx and the slope of Ca2+ spiking were also increased in Scn1a+/− astrocytes. These data indicate that perturbed Ca2+ dynamics in astrocytes may be involved in the pathogenesis of DS.

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Regulated Intramembrane Proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b

Journal of Biological Chemistry ◽

10.1074/jbc.m706661200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1644-1652 ◽

Cited By ~ 96

Author(s):

Lucas Martin ◽

Regina Fluhrer ◽

Karina Reiss ◽

Elisabeth Kremmer ◽

Paul Saftig ◽

...

Keyword(s):

Transmembrane Protein ◽

Loss Of Function ◽

Complex Type ◽

Intramembrane Proteolysis ◽

Type Iv ◽

Regulated Intramembrane Proteolysis ◽

Terminal Fragment ◽

The Family ◽

Membrane Bound

Presenilin, the catalytic component of the γ-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to γ-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to γ-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.

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NBI-921352, a First-in-Class, NaV1.6 Selective, Sodium Channel Inhibitor That Prevents Seizures in Scn8a Gain-of-Function Mice, and Wild-Type Mice and Rats

10.1101/2021.08.29.458125 ◽

2021 ◽

Author(s):

JP Johnson ◽

Thilo Focken ◽

Kuldip Khakh ◽

Parisa Karimi Tari ◽

Celine Dube ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Current ◽

Plasma Concentrations ◽

Wild Type Mouse ◽

State Dependence ◽

Epileptic Encephalopathy ◽

Wild Type ◽

Action Potential Firing ◽

Gain Of Function ◽

Seizure Models

AbstractNBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Moller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC50 0.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756X for NaV1.1, 134X for NaV1.2, 276X for NaV1.7, and >583X for NaV1.3, NaV1.4, and NaV1.5). NBI-921352 is a state-dependent inhibitor, preferentially inhibiting activated (inactivated or open) channels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse, as well as in wild-type mouse and rat seizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor antiseizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 was well tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

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Abstract 2968: Postmortem Genetic Testing of the GPD1-L-Encoded Glycerol-3-Phosphate Dehydrogenase 1-Like Protein in Sudden Infant Death Syndrome

Circulation ◽

10.1161/circ.116.suppl_16.ii_663-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

David W Van Norstrand ◽

Carmen R Valdivia ◽

David J Tester ◽

Kazuo Ueda ◽

Barry London ◽

...

Keyword(s):

Sodium Channel ◽

Sudden Infant Death Syndrome ◽

Current Density ◽

Infant Death ◽

Sodium Current ◽

White Male ◽

Sudden Infant Death ◽

Sudden Infant ◽

Loss Of Function ◽

Glycerol 3 Phosphate Dehydrogenase

Introduction: Approximately 10 – 15% of sudden infant death syndrome (SIDS) may be caused by cardiac channelopathies including Brugada syndrome (BrS). Type 1 BrS (BrS1), due to mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) disrupted trafficking of SCN5A in a multi-generational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of SIDS. Methods: Using DHPLC and direct DNA sequencing, we performed comprehensive open reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 228 anonymous cases of SIDS (86 females, 142 males, average age = 3 + 2 months, range 6 hours to 12 months). Results: Three putative, SIDS-associated GPD1-L missense mutations, E83K, I124V, and R273C, were discovered in a three-month-old white male, a five-week-old white female, and a one-month-old white male, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared to wild type GPD1-L, GPD1-L mutations co-expressed with SCN5A in heterologous HEK cells produced a 60% reduction in peak sodium current density (p<0.004). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current as well (p<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. Conclusion: This study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset (~1%) of SIDS via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal, BrS-like pro-arrhythmic substrate.

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Abstract 356: Loss of Function Mutations of Sodium Channel Beta-1 and Beta-2 Subunits Associated with Atrial Fibrillation and ST-segment Elevation

Circulation ◽

10.1161/circ.116.suppl_16.ii_54-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hiroshi Watanabe ◽

Dawood Darbar ◽

Christiana R Ingram ◽

Kim Jiramongkolchai ◽

Sameer S Chopra ◽

...

Keyword(s):

Atrial Fibrillation ◽

Steady State ◽

Sodium Channel ◽

Sodium Current ◽

Β Subunit ◽

Loss Of Function ◽

St Segment Elevation ◽

Negative Shift ◽

St Segment ◽

Cardiac Sodium Channel

Background: We have recently reported mutations in the cardiac sodium channel gene SCN5A in 5.9% of patients with atrial fibrillation (AF). In this study, we tested the hypothesis that mutations in sodium channel β subunit genes SCN1B-4B contribute to AF susceptibility. Methods and results: All 4 βsubunit genes were resequenced in 376 patients with AF (118 patients with lone AF and 258 patients with AF and cardiovascular disease) and 188 ethnically-defined controls. We identified 2 non-synonymous variants in SCN1B (resulting in R85H, D153N) and 2 in SCN2B (R28Q, R28W) in patients with AF; these occur at residues highly conserved across mammals and were absent in controls. In 3 of 4 mutation carriers, there was saddle back type ST-segment elevation in the right precordial leads of electrocardiogram. Transcripts encoding both SCN1B and SCN2B were detected in human atrium and ventricle. To assess function in vitro , CHO cells were transfected with SCN5A without β subunit, SCN5A with wild-type (WT) β subunit, or SCN5A with mutant β subunit: all 4 mutants altered SCN5A current to a variable extent compared to WT β subunits. WT β1 increased SCN5A currents by 75%, and induced a negative shift in steady-state activation (−10.2 mV) and inactivation (−6.7 mV), compared to SCN5A alone. D153N β1 caused partial loss of function, with increased SCN5A current but to a smaller extent (24%) than WT β1, and a negative shift in steady-state activation (−12.1 mV) and inactivation (−8.1 mV) similar to WT. R85H β1 produced a pure loss of function, with currents no different from SCN5A alone. WT β2 did not change SCN5A current amplitude, while R28Q β2 and R28W β2 decreased current by 36% and 30%, respectively; and positively shifted steady-state activation by +7.4 mV and +5.1 mV, respectively, compared to WT. Conclusion: Loss of function mutations in sodium channel β subunits were identified in patients with AF, and were associated with a distinctive ECG phenotype. These findings further support the hypothesis that decreased sodium current enhances AF susceptibility.

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Sodium channel β1 subunits participate in regulated intramembrane proteolysis-excitation coupling

JCI Insight ◽

10.1172/jci.insight.141776 ◽

2021 ◽

Author(s):

Alexandra A. Bouza ◽

Nnamdi Edokobi ◽

Samantha L. Hodges ◽

Alexa M. Pinsky ◽

James Offord ◽

...

Keyword(s):

Sodium Channel ◽

Intramembrane Proteolysis ◽

Regulated Intramembrane Proteolysis

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Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

10.1101/2020.06.02.129288 ◽

2020 ◽

Author(s):

Zizun Wang ◽

Sarah H. Vermij ◽

Valentin Sottas ◽

Anna Shestak ◽

Daniela Ross-Kaschitza ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Current ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Loss Of Function ◽

Whole Cell ◽

Calmodulin Binding ◽

Neonatal Cardiomyocytes ◽

Terminal Domain ◽

Cardiac Sodium Channel

ABSTRACTThe cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 N-terminal domain (NTD): Y87C–here newly identified–, R104W and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner.Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel with the ER however did not change in the presence of variants, shown by transfected and stained rat neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site.In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.

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