Do sodium channel proteolytic fragments regulate sodium channel expression?

ABSTRACT

The cardiac voltage-gated sodium channel (gene: SCN5A, protein: Na_V1.5) is responsible for the sodium current that initiates the cardiomyocyte action potential. Research into the mechanisms of SCN5A gene expression has gained momentum over the last few years. We have recently described the transcriptional regulation of SCN5A by GATA4 transcription factor. In this addendum to our study, we report our observations that 1) the linker between domains I and II (L_DI-DII) of Na_V1.5 contains a nuclear localization signal (residues 474–481) that is necessary to localize L_DI-DII into the nucleus, and 2) nuclear L_DI-DII activates the SCN5A promoter in gene reporter assays using cardiac-like H9c2 cells. Given that voltage-gated sodium channels are known targets of proteases such as calpain, we speculate that Na_V1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of L_DI-DII and subsequent stimulation of the SCN5A promoter.

Introduction

Voltage-gated sodium channels are vital proteins in cardiac physiology. Upon changes in membrane potential, sodium channels open and enable the inward, depolarising sodium currents that underlie Phase 0 of the cardiomyocyte action potential.¹ Na_V1.5, encoded by the SCN5A gene, is the pore-forming, α subunit, of the cardiac voltage-gated sodium channel, and is necessary and sufficient to generate voltage-dependent, inward sodium currents. Na_V1.5 is an essential protein and Scn5A −/− mice are not viable, while Scn5A +/− knockout mice display cardiac conduction defects and ventricular tachycardia.²

In humans, genetic variants in SCN5A have been linked to cardiac arrhythmias (atrial and ventricular fibrillation), sudden cardiac death syndromes (Brugada syndrome, long QT syndrome, sudden infant death syndrome) and other cardiac phenotypes (conduction defects, sick sinus syndrome).³ We have recently shown that SCN5A expression is regulated by the GATA4 transcription factor in the human heart.⁴ Recent evidence suggests that abnormal SCN5A gene expression is associated with arrhythmogenic diseases (discussed in Tarradas et al. 2017),⁴ and therefore the understanding of how SCN5A expression is controlled constitutes an important step forward in the field of cardiac diseases related to Na_V1.5 dysfunction.

Na_V1.5 is a large (2016 residues, ca. 220 KDa), hydrophobic, integral membrane protein that consists of 4 homologous domains (termed DI to DIV), joined by cytosolic interdomain linkers.¹ Great efforts over the past 7 y have shed light onto the structure of other voltage-gated sodium channels (mainly bacterial proteins),^5,6 and the first structure of an eukaryotic voltage-gated sodium channel α subunit has recently been solved.⁷ While these and other studies have provided invaluable insight into voltage-gating mechanisms and pore structure, the role and organization of cytosolic domains is less clear. The linker between domains DI and DII (L_DI-DII) of Na_V1.5 contains 295 residues and has been of particular interest to us and other groups due to the fact that L_DI-DII undergoes post-translational modifications including phosphorylation and arginine methylation (for a recent review see⁸). Biochemical, genetic and electrophysiological studies suggest that L_DI-DII participates in the regulation of channel inactivation.⁹

Voltage-gated sodium channels have long been known to be regulated by proteases.¹⁰ For example, calpain cleaves the brain isoform of the voltage-gated sodium channel, Na_V1.2, at L_DI-DII.¹¹ Calpain sodium channel fragments interact and localize at the plasma membrane hours after calpain activation,¹¹ suggesting that these fragments retain the protein–protein interactions that hold the sodium channel macromolecular complex together. However, there is currently no information on whether these fragments are subsequently degraded. A thought-provoking alternative is that sodium channel proteolysis creates new proteins with modified biologic activities. In this addendum, we further our understanding of SCN5A gene expression by showing that L_DI-DII contains a nuclear localization signal (NLS), localizes to the cell nucleus when expressed as an isolated domain in cardiac-like H9c2 cells, and increases SCN5A promoter activity in vitro.

Results

L_DI-DII is predicted to be a target for calpain and contains an NLS

Previous reports have shown that L_DI-DII from Na_V1.2 is a target for calpain. We used 2 published protease site prediction algorithms to search for possible calpain cleavage sites in the Na_V1.5 L_DI-DII sequence (residues 416–711).^12,13 Both algorithms predicted calpain cleavage after position E462. The other common hot spots for calpain cleavage in L_DI-DII were the regions spanning residues 512–524, 573–579, and 630–644 (Fig. 1A).

Figure 1.

(A) Sequence of L_DI-II (residues 416–711 of Na_V1.5) highlighting predicted calpain cleavage sites (E462, and regions 512–524, 573–579, and 630–644) as well as the putative NLS (underlined). (B) Sequence alignment of possible NLS in Na_V members (Na_V1.5 numbering), highlighting conserved arginines (in black) and R residues 474, 475, 478, 479 and 481 (underlined).

Inspection of the sodium channel L_DI-DII sequences revealed the presence of a conserved arginine-rich motif including residues 474–481 (Fig. 1A and B). We hypothesized that this motif could be a classical basic NLS. To test this hypothesis, we cloned the entire Na_V1.5 L_DI-DII as a FLAG-YFP fusion protein in a mammalian expression vector. We transfected this plasmid into H9c2 cells and observed L_DI-DII expression mainly in the nucleus (Fig. 2, left). When we mutated R474, R475, R478, R479 and R481 to K residues, the localization of the linker was no longer restricted to cell nuclei (Fig. 2, right). We repeated these transfections more than 10 times and reproducibly observed a similar fluorescence pattern.

Figure 2.

Representative confocal microscopy images of H9c2 cells transfected with L_DI-II including intact (left) and mutated (right) NLS. Scale bar corresponds to 50 µm.

L_DI-DII increases the activity of SCN5A promoter

Why would an isolated cytosolic domain of a voltage-gated sodium channel localize to the cell nucleus? We asked whether L_DI-DII could modify SCN5A expression. We transfected H9c2 cells with a plasmid expressing luciferase under the control of the SCN5A promoter (“promoter A” in the original publication),⁴ together with plasmids expressing either L_DI-DII with an intact NLS, or L_DI-DII with a mutated NLS (R474, R475, R478, R479 and R481 to K as above). Our luciferase assays showed that L_DI-DII with intact NLS, but not the mutated version, stimulated SCN5A promoter activity in a dose-dependent manner (Fig. 3).

Figure 3.

(A) Luciferase experiments in H9c2 cells transfected with an SCN5A promoter-luciferase construct and increasing amounts (447 and 895 ng) of the indicated pcDNA3.1-based L_DI-II expression plasmids encoding for L_DI-II with intact and mutated NLS (residues 474–481). Luciferase values were normalized to renilla and are shown as fold induction relative to non-overexpressing control conditions (mean ± SEM, n = 4). Significance was examined by the t-test relative to control: * p = 0.03, ** p = 0.001. (B) Representative Western blot showing expression of L_DI-II with intact and mutated NLS (895 ng plasmid each).

Discussion

In this addendum, we have built on our recently published investigation of SCN5A transcriptional regulation,⁴ to identify another possible mechanism controlling SCN5A gene expression i. e. L_DI-DII stimulation of the SCN5A proximal promoter. We have identified an NLS in L_DI-DII and provided evidence that nuclear L_DI-DII enhances SCN5A transcription. These new results should be regarded as preliminary at this stage given the nature of our cell and in vitro studies, but they raise the exciting possibility that SCN5A expression may be regulated by sodium channel proteolysis.

Proteolysis of sodium channel β subunits is known to regulate transcription of α subunits such as Na_V1.1, the brain isoform of the voltage-gated sodium channel. The group of Kovacs, and others, demonstrated that 1) ADAM10 and BACE1 proteases cleave off the extracellular domain of the β2 subunit; 2) γ-secretase releases the β2 intracellular domain, and 3) the β2 intracellular domain is internalised into the cell nucleus (by unknown mechanisms) and induces an increase in Na_V1.1 mRNA and protein levels.^14,15,16 The β1 subunit is also a target for BACE1 in vitro,¹⁶ and β1 silencing has recently been shown to result in decreased Na_V1.1 mRNA (and protein) levels in cell models.¹⁷

NLS often consist of short arginine-rich sequences, and have been described in voltage-gated potassium channels, notably K_V10.1.¹⁸ We have found that Na_V1.5 L_DI-DII localizes to the cell nucleus when expressed as an isolated domain in H9c2 cells, and that this nuclear localization is abrogated by mutation of 5 R residues to K in the sequence RRSKRRKR spanning residues 474–481 of Na_V1.5. To our knowledge, this is the first report of an NLS in α subunits of voltage-gated sodium channels. Sequence alignment suggests that the NLS could be conserved among other members of the voltage-gated sodium channel family, including conservation of R474 and R475 in 4 and 5 members of the family, respectively, and an essential R478 (Na_V1.5 numbering throughout, Fig. 1B). Taking together our new observations with previous reports, it is tempting to speculate that calpain (alone or in combination with other proteases) cleavage of α subunits^11,19 creates L_DI-DII fragments containing NLS, and that nuclear L_DI-DII play a role in cardiac sodium channel transcriptional regulation, likely in combination with transcription factors.

Our approach was limited by the lack of information on precise calpain proteolytic site(s) within L_DI-DII. Both algorithms used here predicted cleavage after E462, and we identified 3 possible calpain sites C-terminal to the NLS. Given these uncertainties, we decided to clone the complete 295-residue long L_DI-DII domain and not smaller L_DI-DII fragments. Mapping both calpain sites on L_DI-DII experimentally and identifying the required sequences in L_DI-DII necessary for activation of SCN5A transcription would greatly help design further experiments to understand the relevance of our results in more physiologic contexts. While acknowledging these weaknesses, this report raises the intriguing possibility that mutations and post-translational modifications of L_DI-DII may control sodium channel activity not only at the electrophysiological level but also at the gene expression level. Bearing in mind that 1) the exact role of L_DI-DII remains to be fully explored, 2) there are at least 63 disease-causing mutations in L_DI-DII,²⁰ and 3) there are 15–20 phosphorylation sites,^21,22 as well as 3 arginine methylation sites (R513, R526 and R680)⁹ in L_DI-DII, our findings provide a new lens to look at the involvement of L_DI-DII in Na_V1.5 currents and arrhythmogenic diseases.

Methods

Cells and plasmids

Cardiac cells derived from embryonic rat ventricle (H9c2 cells) were maintained under standard cell culture conditions. L_DI-DII (residues 416–711) was amplified by PCR from a pCDNA3.1-based plasmid encoding for Na_V1.5,⁹ and cloned as a FLAG-YFP fusion into pCDNA3.1.²³ Site-directed mutagenesis was done using the Quikchange kit from Agilent Technologies, following the instructions of the manufacturer (http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange-Lightning/?cid = AG-PT-175andtabId = AG-PR-1162). Sanger sequencing was performed in-house to ensure the introduction of the desired mutations. Cells were transfected using Lipofectamine 2000 (Thermo Fisher, https://www.thermofisher.com/uk/en/home/brands/product-brand/lipofectamine/lipofectamine-2000.html) and analyzed 24–48 h after transfection.

Prediction of calpain sites in L_DI-DII

We used recently published algorithms to predict calpain sites in L_DI-DII. The FASTA sequence of L_DI-DII was analyzed using the CaMPDB and GPS-CCD databases,^12,13 at score thresholds of 0.14 and 0.9, respectively.

Confocal microscopy

Cells were fixed (4% paraformaldehyde) and permeabilised in 1% Triton-X100. We used an anti-FLAG antibody to detect L_DI-DII (Sigma, http://www.sigmaaldrich.com/catalog/product/sigma/f3165?lang = enandregion = GB), as well as fluorescent phalloidin to mark F-actin (Thermo Fisher, https://www.thermofisher.com/order/catalog/product/A12379) and DAPI for nuclear staining. Cells were visualised by confocal microscopy in a Zeiss LSM 710.

Reporter assays

Luciferase / Renilla assays were performed as described in the original publication,⁴ using L_DI-DII plasmids. Results from 4 biologic replicates are reported here.

Western blot

H9c2 cells were lysed in 1% NP-40 48 h after transfection. We used an anti-GFP antibody (Abcam, http://www.abcam.com/gfp-antibody-chip-grade-ab290.html) to detect L_DI-DII-FLAG-YFP expression. Two biologic replicates were done.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Kath Bulmer for excellent technical assistance. The authors thank students who over the years have contributed to the present work, including David Roura and Oliver Taylor.

Funding

DOO acknowledges Rivers state university of Science & Technology Port Harcourt Nigeria, and TETfund Nigeria (Academic staff Training and development unit) for funding. AT acknowledges a predoctoral fellowship from University of Girona (BR2012/47), MP-A a predoctoral fellowship from Generalitat de Catalunya (2014FI_B 00586). This work was supported by the University of Hull, the Spanish Government (SAF2011–27627) and 7PM-PEOPLE Marie Curie International Reintegration Grant (PIRG07-GA-2010–268395).