Investigations of the Navβ1b sodium channel subunit in human ventricle; functional characterization of the H162P Brugada syndrome mutant

Brugada syndrome (BrS) is a rare inherited disease that can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Nav1.5 have been associated with BrS. However, few mutations in the auxiliary Navβ1–4 subunits have been linked to this disease. Here we investigated differences in expression and function between Navβ1 and Navβ1b and whether the H162P/Navβ1b mutation found in a BrS patient is likely to be the underlying cause of disease. The impact of Navβ subunits was investigated by patch-clamp electrophysiology, and the obtained in vitro values were used for subsequent in silico modeling. We found that Navβ1b transcripts were expressed at higher levels than Navβ1 transcripts in the human heart. Navβ1 and Navβ1b coexpressed with Nav1.5 induced a negative shift on steady state of activation and inactivation compared with Nav1.5 alone. Furthermore, Navβ1b was found to increase the current level when coexpressed with Nav1.5, Navβ1b/H162P mutated subunit peak current density was reduced by 48% (−645 ± 151 vs. −334 ± 71 pA/pF), V1/2 steady-state inactivation shifted by −6.7 mV (−70.3 ± 1.5 vs. −77.0 ± 2.8 mV), and time-dependent recovery from inactivation slowed by >50% compared with coexpression with Navβ1b wild type. Computer simulations revealed that these electrophysiological changes resulted in a reduction in both action potential amplitude and maximum upstroke velocity. The experimental data thereby indicate that Navβ1b/H162P results in reduced sodium channel activity functionally affecting the ventricular action potential. This result is an important replication to support the notion that BrS can be linked to the function of Navβ1b and is associated with loss-of-function of the cardiac sodium channel.

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P1597Two novel mutations in SCN5A gene cause enhanced inactivation and lead to Brugada syndrome

European Heart Journal ◽

10.1093/eurheartj/ehz748.0356 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

A Zaytseva ◽

A V Karpushev ◽

Y Fomicheva ◽

...

Keyword(s):

Steady State ◽

Sodium Channel ◽

Brugada Syndrome ◽

Slow Inactivation ◽

Fast Inactivation ◽

Novel Mutations ◽

Patch Clamp Technique ◽

Inactivation Curve ◽

Voltage Gated ◽

Cardiac Sodium Channel

Abstract Background Mutations in gene SCN5A, encoding cardiac potential-dependent sodium channel Nav1.5, are associated with various arrhythmogenic disorders among which the Brugada syndrome (BrS) and the Long QT syndrome (LQT) are the best characterized. BrS1 is associated with sodium channel dysfunction, which can be reflected by decreased current, impaired activation and enhanced inactivation. We found two novel mutations in our patients with BrS and explored their effect on fast and slow inactivation of cardiac sodium channel. Purpose The aim of this study was to investigate the effect of BrS (Y739D, L1582P) mutations on different inactivation processes in in vitro model. Methods Y739D and L1582P substitutions were introduced in SCN5A cDNA using site-directed mutagenesis. Sodium currents were recorded at room temperature in transfected HEK293-T cells using patch-clamp technique with holding potential −100 mV. In order to access the fast steady-state inactivation curve we used double-pulse protocol with 10 ms prepulses. To analyze voltage-dependence of slow inactivation we used two-pulse protocol with 10s prepulse, 20ms test pulse and 25ms interpulse at −100mV to allow recovery from fast inactivation. Electrophysiological measurements are presented as mean ±SEM. Results Y739D mutation affects highly conserved tyrosine 739 among voltage-gated sodium and calcium channels in the segment IIS2. Mutation L1582P located in the loop IVS4-S5, and leucine in this position is not conserved among voltage-gated channels superfamily. We have shown that Y739D leads to significant changes in both fast and slow inactivation, whereas L1582P enhanced slow inactivation only. Steady-state fast inactivation for Y739D was shifted on 8.9 mV towards more negative potentials compare with that for WT, while L1582P did not enhanced fast inactivation (V1/2 WT: −62.8±1.7 mV; Y739D: −71.7±2.3 mV; L1582P: −58.7±1.4 mV). Slow inactivation was increased for both substitutions (INa (+20mV)/INa (−100mV) WT: 0.45±0.03; Y739D: 0,34±0.09: L1582P: 0.38±0.04). Steady-state fast inactivation Conclusions Both mutations, observed in patients with Brugada syndrome, influence on the slow inactivation process. Enhanced fast inactivation was shown only for Y739D mutant. The more dramatic alterations in sodium channel biophysical characteristics are likely linked with mutated residue conservativity. Acknowledgement/Funding RSF #17-15-01292

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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 current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2017.10.002 ◽

2018 ◽

Vol 114 ◽

pp. 10-19 ◽

Cited By ~ 18

Author(s):

Elisabet Selga ◽

Franziska Sendfeld ◽

Rebecca Martinez-Moreno ◽

Claire N. Medine ◽

Olga Tura-Ceide ◽

...

Keyword(s):

Stem Cell ◽

Sodium Channel ◽

Brugada Syndrome ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Syndrome Patient ◽

Loss Of Function ◽

Current Loss ◽

Channel Current ◽

Induced Pluripotent

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Differential effects of cardiac sodium channel mutations on initiation of ventricular arrhythmias in patients with Brugada syndrome

Heart Rhythm ◽

10.1016/j.hrthm.2009.01.031 ◽

2009 ◽

Vol 6 (4) ◽

pp. 487-492 ◽

Cited By ~ 18

Author(s):

Hiroshi Morita ◽

Satoshi Nagase ◽

Daiji Miura ◽

Aya Miura ◽

Shigeki Hiramatsu ◽

...

Keyword(s):

Sodium Channel ◽

Brugada Syndrome ◽

Ventricular Arrhythmias ◽

Differential Effects ◽

Cardiac Sodium Channel

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A cardiac sodium channel mutation identified in Brugada syndrome associated with atrial standstill

Journal of Internal Medicine ◽

10.1046/j.0954-6820.2003.01247.x ◽

2004 ◽

Vol 255 (1) ◽

pp. 137-142 ◽

Cited By ~ 62

Author(s):

N. Takehara ◽

N. Makita ◽

J. Kawabe ◽

N. Sato ◽

Y. Kawamura ◽

...

Keyword(s):

Sodium Channel ◽

Brugada Syndrome ◽

Cardiac Sodium Channel ◽

Channel Mutation ◽

Sodium Channel Mutation

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Functional characterization of a loss-of-function mutant I324M of arginine vasopressin receptor 2 in X-linked nephrogenic diabetes insipidus

Scientific Reports ◽

10.1038/s41598-021-90736-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lixia Wang ◽

Weihong Guo ◽

Chunyun Fang ◽

Wenli Feng ◽

Yumeng Huang ◽

...

Keyword(s):

Diabetes Insipidus ◽

Arginine Vasopressin ◽

Nephrogenic Diabetes Insipidus ◽

Functional Characterization ◽

Gene Mutations ◽

Biochemical Properties ◽

Vasopressin Receptor ◽

Inherited Disease ◽

Loss Of Function ◽

Gene Encoding

AbstractX-linked nephrogenic diabetes insipidus (X-linked NDI) is a rare inherited disease mainly caused by lost-of-function mutations in human AVPR2 gene encoding arginine vasopressin receptor 2 (V2R). Our focus of the current study is on exploration of the functional and biochemical properties of Ile324Met (I324M) mutation identified in a pedigree showing as typical recessive X-linked NDI. We demonstrated that I324M mutation interfered with the conformation of complex glycosylation of V2R. Moreover, almost all of the I324M-V2R failed to express on the cell surface due to being captured by the endoplasmic reticulum control system. We further examined the signaling activity of DDAVP-medicated cAMP and ERK1/2 pathways and the results revealed that the mutant receptor lost the ability in response to DDAVP stimulation contributed to the failure of accumulation of cAMP and phosphorylated ERK1/2. Based on the characteristics of molecular defects of I324M mutant, we selected two reagents (SR49059 and alvespimycin) to determine whether the functions of I324M-V2R can be restored and we found that both compounds can significantly “rescue” I324M mutation. Our findings may provide further insights for understanding the pathogenic mechanism of AVPR2 gene mutations and may offer some implications on development of promising treatments for patients with X-linked NDI.

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Sodium channel kinetic changes that produce Brugada syndrome or progressive cardiac conduction system disease

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01025.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. H399-H407 ◽

Cited By ~ 33

Author(s):

Zhu-Shan Zhang ◽

Joseph Tranquillo ◽

Valentina Neplioueva ◽

Nenad Bursac ◽

Augustus O. Grant

Keyword(s):

Action Potential ◽

Sodium Channel ◽

Brugada Syndrome ◽

Sodium Current ◽

Conduction System ◽

Cardiac Conduction ◽

Cardiac Conduction System ◽

Wild Type ◽

System Disease ◽

Conduction System Disease

Some mutations of the sodium channel gene NaV1.5 are multifunctional, causing combinations of LQTS, Brugada syndrome and progressive cardiac conduction system disease (PCCD). The combination of Brugada syndrome and PCCD is uncommon, although they both result from a reduction in the sodium current. We hypothesize that slow conduction is sufficient to cause S-T segment elevation and undertook a combined experimental and theoretical study to determine whether conduction slowing alone can produce the Brugada phenotype. Deletion of lysine 1479 in one of two positively charged clusters in the III/IV inter-domain linker causes both syndromes. We have examined the functional effects of this mutation using heterologous expression of the wild-type and mutant sodium channel in HEK-293-EBNA cells. We show that ΔK1479 shifts the potential of half-activation, V1/2m, to more positive potentials ( V1/2m = −36.8 ± 0.8 and −24.5 ± 1.3 mV for the wild-type and ΔK1479 mutant respectively, n = 11, 10). The depolarizing shift increases the extent of depolarization required for activation. The potential of half-inactivation, V1/2h, is also shifted to more positive potentials ( V1/2h = −85 ± 1.1 and −79.4 ± 1.2 mV for wild-type and ΔK1479 mutant respectively), increasing the fraction of channels available for activation. These shifts are quantitatively the same as a mutation that produces PCCD only, G514C. We incorporated experimentally derived parameters into a model of the cardiac action potential and its propagation in a one dimensional cable (simulating endo-, mid-myocardial and epicardial regions). The simulations show that action potential and ECG changes consistent with Brugada syndrome may result from conduction slowing alone; marked repolarization heterogeneity is not required. The findings also suggest how Brugada syndrome and PCCD which both result from loss of sodium channel function are sometimes present alone and at other times in combination.

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Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

Biochemical Journal ◽

10.1042/bj20051606 ◽

2006 ◽

Vol 394 (1) ◽

pp. 163-171 ◽

Cited By ~ 11

Author(s):

Sandra Müller ◽

Jennifer Disse ◽

Manuela Schöttler ◽

Sylvia Schön ◽

Christian Prante ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Activity ◽

Binding Capacity ◽

Functional Characterization ◽

Terminal Region ◽

Binding Motif ◽

Heparin Binding ◽

Loss Of Function ◽

The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

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Abstract 20627: Modulation of Cardiac Sodium Channel by Palmitoylation and Its Association With Cardiac Disease Mutations

Circulation ◽

10.1161/circ.130.suppl_2.20627 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Zifan Pei ◽

Andy Hudmon ◽

Theodore R Cummins

Keyword(s):

Steady State ◽

Sodium Channel ◽

Functional Activity ◽

Site Directed Mutagenesis ◽

Significant Shift ◽

Biophysical Properties ◽

Disease Mutations ◽

Cardiac Sodium Channel ◽

Steady State Inactivation

Cardiac sodium channel (Nav1.5) is responsible for the generation and propagation of the cardiac action potential, which underlies cardiac excitability. It can be modified by a variety of post-translational modifications. Palmitoylation is one of the most common post-translational lipid modifications that can dynamically regulate protein life cycle and functional activity. In our study, we identified palmitoylation on Nav1.5 and its alteration in channel biophysical properties. Nav1.5 palmitoylation was identified in both HEK 293 cells stably expressing Nav1.5 and cardiac tissues using acyl-biotin exchange assay. Nav1.5 palmitoylation was inhibited by pre-incubating the cells with the inhibitor 2-Br-Palmitate (2BP, 25uM, 24hrs). Biophysically, 2BP treatment drastically shifted the channel steady-state inactivation to more hyperpolarized voltages, suggesting palmitoylation altering channel functional activity. In addition, four predicted endogenous palmitoylation sites were identified using CSS-Palm 3.0. Site-directed mutagenesis method was used to generate a cysteine removing background of wt Nav1.5 to study the role of predicted sites. Patch clamp analysis of wt and cysteine-removed Nav1.5 revealed a significant change in channel biophysics. 2BP treatment significantly shifted steady-state inactivation of wt Nav1.5 while not affecting cysteine-removed Nav1.5 significantly, indicating the important role of these four cysteine sites in modulating channel palmitoylation. Moreover, several LQT disease mutations were identified to potentially add or remove palmitoylation sites. Further analysis of these disease mutations revealed a significant shift in channel steady-state inactivation and this alteration cannot be seen with the substitution of other residues on the same site, suggesting the specific role of cysteine residue in causing the functional alteration. For the LQT mutation that removes potential palmitoylation site, 2BP treatment did not affect channel biophysical properties, indicating the essential role of this cysteine in channel palmitoylation. These results suggest that palmitoylation on Nav1.5 regulates channel functional activity and its modulation may contribute to new cardiac channelopathies.

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