Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications

    Regulation of the cardiac Na + channel NaV 1.5 by post-translational modifications C´eline Marionneau, Hugues Abriel PII: DOI: Reference:

S0022-2828(15)00058-9 doi: 10.1016/j.yjmcc.2015.02.013 YJMCC 8023

To appear in:

Journal of Molecular and Cellular Cardiology

Received date: Revised date: Accepted date:

9 December 2014 28 January 2015 17 February 2015

Please cite this article as: Marionneau C´eline, Abriel Hugues, Regulation of the cardiac Na+ channel NaV 1.5 by post-translational modifications, Journal of Molecular and Cellular Cardiology (2015), doi: 10.1016/j.yjmcc.2015.02.013

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Submitted to JMCC

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Review Article

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Regulation of the cardiac Na+ channel NaV1.5 by post-translational

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modifications

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Céline Marionneau1 & Hugues Abriel2 1

Department of Clinical Research, University of Bern, Bern, Switzerland

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L'institut du thorax; Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1087; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6291; Université de Nantes, Nantes, France

Correspondence to:

Dr. Céline Marionneau, PhD L'institut du thorax, INSERM UMR1087, CNRS UMR6291 IRS-Université de Nantes, 8 Quai Moncousu, BP 70721 44007 Nantes Cedex 1, France Phone: 33-2-28-08-01-63 Fax: 33-2-28-08-01-30 Email: [email protected] or Dr. Hugues Abriel, MD PhD University of Bern, Department of Clinical Research Murtenstrasse 35, 3010 Bern, Switzerland Phone: 41-31-6320928 Fax: 41-31-6320946 Email: [email protected]

ACCEPTED MANUSCRIPT Abstract The cardiac voltage-gated Na+ channel, NaV1.5, is responsible for the upstroke of the action potential in cardiomyocytes and for efficient propagation of the electrical impulse in the myocardium. Even subtle alterations of NaV1.5 function, as caused by mutations in its gene SCN5A, may lead to

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many different arrhythmic phenotypes in carrier patients. In addition, acquired malfunctions of NaV1.5

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that are secondary to cardiac disorders such as heart failure and cardiomyopathies, may also play significant roles in arrhythmogenesis. While it is clear that the regulation of NaV1.5 protein expression

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and function tightly depends on genetic mechanisms, recent studies have demonstrated that NaV1.5 is the target of various post-translational modifications that are pivotal not only in physiological

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conditions, but also in disease. In this review, we examine the recent literature demonstrating glycosylation, phosphorylation by Protein Kinases A and C, Ca2+/Calmodulin-dependent protein

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Kinase II, Phosphatidylinositol 3-Kinase, Serum- and Glucocorticoid-inducible Kinases, Fyn and Adenosine Monophosphate-activated Protein Kinase, methylation, acetylation, redox modifications, and ubiquitylation of NaV1.5. Modern and sensitive mass spectrometry approaches, applied directly to

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channel proteins that were purified from native cardiac tissues, have enabled the determination of the

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precise location of post-translational modification sites, thus providing essential information for understanding the mechanistic details of these regulations. The current challenge is first, to understand

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the roles of these modifications on the expression and the function of NaV1.5, and second, to further identify other chemical modifications. It is postulated that the diversity of phenotypes observed with NaV1.5-dependent disorders may partially arise from the complex post-translational modifications of channel protein components.

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ACCEPTED MANUSCRIPT Keywords: Cardiac NaV1.5 channels; Post-translational modifications; Arrhythmias; Native proteomics

Abbreviations

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Ac: Acetylated

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AMPK: Adenosine Monophosphate-activated Protein Kinase BrS: Brugada Syndrome

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CaMKII: Ca2+/Calmodulin-dependent Kinase II INa: Cardiac voltage-gated Na+ current

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INaL: Cardiac voltage-gated late Na+ current LQTS: Long QT syndrome

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Me: Methylated MS: Mass Spectrometry

PI3K: Phosphatidylinositol 3-Kinase

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NaV  subunit: Voltage-gated Na+ (NaV) channel pore-forming () subunit

PKB/Akt: Protein Kinase B

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PKC: Protein Kinase C

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PKA: cAMP-dependent Protein Kinase

pS: Phosphoserine

pT: Phosphothreonine

PTM: Post-translational Modification pY: Phosphotyrosine

Redox: Reduction/oxidation

SGK: Serum- and Glucocorticoid-inducible Kinase

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ACCEPTED MANUSCRIPT 1.

Introduction The action potential of most cardiac cells and the propagation of the electrical impulse through

cardiac tissue depend essentially on the cardiac Na+ current (INa) [1]. Cardiac INa is mainly mediated by

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the voltage-gated Na+ (NaV) channel, NaV1.5 [2]. Other isoforms of the NaV channel subfamily have

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also been suggested to be expressed in cardiomyocytes, albeit at a lower level of expression [3]. Note

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that the role of these "non-cardiac" NaV channels in cardiomyocytes is currently poorly understood and debated. The role of NaV1.5 in inherited and acquired cardiac disorders has been the object of intense

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research over the past twenty years [2, 4, 5]. One of the most striking observations is that hundreds of

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genetic variants in SCN5A, the gene located on chromosome 3p21 that codes for NaV1.5, have been found in patients and families with inherited forms of cardiac arrhythmias and cardiomyopathies [5, 6].

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In terms of their prevalence, these SCN5A mutations are mainly found in patients with two phenotypes,

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i.e. congenital long QT syndrome (LQTS) type 3 and Brugada syndrome (BrS) [7]. In most cases, these

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mutations lead to either an increase in late Na+ current (INaL) when associated with LQTS, or a loss of function caused by diverse mechanisms when found in BrS patients. The long list of cardiac pathological phenotypes, found in patients with mutations in SCN5A, remains a puzzling question [4, 5] and may suggest that, besides its well-described role in cardiac excitability and conduction, NaV1.5 may also play other non-canonical roles in cardiomyocytes and other cell types [8]. The whole cardiac Na+ channel is composed of the NaV1.5  subunit that is the pore-forming protein and associated proteins [2, 9]. The NaV1.5 protein has a molecular weight of about 220 kDa and the main adult cardiac human splice variant, hH1C, comprises 2015 amino acids [10]. NaV1.5 has been shown to assemble with small (about 30-40 kDa), single transmembrane segment proteins called β subunits [11]; four of these β subunits have been found to be encoded in the human genome. NaV1.5 is composed of cytoplasmic N- and C-terminal domains, as well as four homologous domains (DI-DIV), each consisting of six transmembrane segments S1 to S6 (Figure 1). The first four transmembrane 4

ACCEPTED MANUSCRIPT segments (S1-S4) comprise the voltage-sensing domain, and the last two segments (S5 and S6) form the ion permeation pathway of the channel when folded in the membrane. The four homologous domains are linked by intracellular loops DI-II, DII-III and DIII-IV. Similar to most membrane

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proteins, NaV1.5 has been found to interact with several different proteins that regulate the expression

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and the function of the channel [2, 9]. These associated proteins are classified as anchoring/adaptor

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proteins, enzymes that interact with and modify the channel structure through post-translational modifications (PTMs), and proteins that modulate the biophysical properties of NaV1.5 upon binding.

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Recent studies demonstrated that NaV1.5 channels are found in at least two distinct membrane pools in

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cardiac cells and that they associate with different proteins depending upon their localization at the intercalated discs or the lateral membranes of the cell (reviewed in [12]).

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Together with associated/regulatory proteins, PTMs of NaV1.5 channels are critical in

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regulating various aspects of cardiac NaV1.5 channel physiology and pathophysiology, relaying signals

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directly from activated intracellular pathways to cardiac excitability [13]. The scope of this review is to give a comprehensive overview of current knowledge with respect to the regulation of cardiac NaV1.5 channels through direct PTMs of the NaV1.5 channel  subunits. With the exception of NaV1 which has been suggested to be N-glycosylated [14], no modifications of associated/regulatory proteins of NaV1.5 have thus far been reported. In particular, we will compare the findings obtained using in silico prediction and/or in vitro analyses with the more recent mass spectrometry (MS)-based proteomic identification of native modifications in channel proteins that were purified from cardiac tissues. Although numerous hypothesis-driven and/or in silico/in vitro studies have suggested, and will certainly continue to suggest many roles for specific modifying enzymes and associated amino acid residues on channel proteins in mediating post-translational regulations, only recently have sophisticated biochemical and proteomic methods become available to unveil specific modification sites and pave the way for the comprehension of underlying mechanistic details. In this regard, Figure 5

ACCEPTED MANUSCRIPT 1 provides a direct comparison of PTM sites that were identified using in silico and/or in vitro analyses with those that were obtained using native proteomics. This particular point is also made in Tables 1 and 2 where we mention whether the presence of identified site(s) (3rd column) and/or the function of

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PTMs (5th column) have been validated in native cardiac cells. In addition to reporting the effects of

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PTMs on regulating the expression and/or the function of cardiac NaV1.5 channels under basal

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physiological conditions, we summarize the pathological relevance of these direct chemical modifications. While altered PTMs of NaV1.5 channels may indeed causally contribute to acquired

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diseases, such as ischemic heart disease and heart failure, mutations in SCN5A or genes encoding

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associated/regulatory proteins could also mimic or abolish these modifications and lead to diseased phenotypes. These pathological implications are discussed in each paragraph below and are listed in the

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6th and 7th columns of Tables 1 and 2. Finally, Figure 2 illustrates the chemical structures of post-

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translationally-modified amino acid residues described for NaV1.5 and/or NaV1.

Advantages and limitations of mass spectrometry-based proteomic identification of native

post-translational modifications

Combined biochemistry and MS-based proteomic analyses are being increasingly exploited to provide unbiased approaches for identifying the native PTMs of ion channels [15-22]. Proteomic workflow typically consists of solubilizing and purifying channel proteins from tissue lysates by immunoprecipitation with specific antibodies, digesting entire immunoprecipitates using endoproteases, and identifying PTMs by liquid chromatography-tandem mass spectrometry (LCMS/MS) [23]. The LC-MS/MS analyses comprise two steps. The first step, which generates the MS1 or MS spectra, allows for measurement of m/z and relative abundance of precursor ion peptides. The most abundant precursor ion peptides are then isolated for fragmentation, and the m/z of fragmented ion peptides are determined in tandem MS spectra (also called fragmentation, MS/MS or MS2 spectra). 6

ACCEPTED MANUSCRIPT Determination of mass differences between fragmented ion peptides provides amino acid sequence information and PTM localization. The application of proteomic approaches for the identification of PTMs has offered several clear advantages, especially over more classical in silico and in vitro

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methods, revealing, without a priori knowledge, novel, unexpected, and importantly, native sites of

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modifications. Different proteomic quantification methods, including the use of label-free

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quantification of high mass resolution MS1 data, also make it possible to compare the relative proportions of various PTMs in different experimental samples with good precision and linearity [24,

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25], which is required to fully characterize a biological response.

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Nevertheless, proteomic approaches also have substantial limitations, mainly with its low sensitivity and the necessity to use large amounts of starting materials. Regulatory PTMs have

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substoichiometric occupancy on their target proteins, making it challenging to achieve sensitivity.

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Likewise, biologically relevant PTMs might occur only under a very restricted set of circumstances, or

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on specific isoforms or species, which might not be part of the conditions used in the proteomic analysis. Moreover, PTMs are labile; in other words, our ability to maintain their integrity during the numerous steps involved in the immunoaffinity purification is difficult, often requiring the development of special enrichment methods. Additionally, the choice of proteases for PTM analysis is crucial, as it determines the length of peptides and therefore our ability to detect them in MS [26]. Finally, low MS sensitivity often results in insufficient peptide fragmentation information, thus making it difficult to assign the site of modification with single amino acid resolution. This is especially true for peptides containing multiple possible sites of modification (i.e., multiple serines, threonines and/or tyrosines in phosphopeptides). As a consequence of all these methodological caveats, it is important in the development of these approaches to keep in mind that the absence of a PTM does not necessarily mean that the PTM is not present. Determination of the relative occupancy or stoichiometry of PTM sites, i.e. the fraction of modified proteins, is also difficult. Another clear limitation of proteomic 7

ACCEPTED MANUSCRIPT analyses is the potential for identified PTMs to occur during lysis and immunoaffinity purification experiments. This limitation emphasizes the need to validate the functional roles of newly identified PTM sites in native cells using alternative experimental approaches.

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Altogether, these methodological limitations must be taken into account and nuance apparent

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discrepancies in the results obtained using in silico/in vitro and native proteomic analyses. It is also

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important to keep in mind that proteomics functions as a first screen in a biology project, and that extracting functional knowledge from the data can be nontrivial. Proteomics reveals a myriad of

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modifications, and physiologists are faced with several challenges: how to select a small number of

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sites to study; how to identify the modifying enzyme(s) targeting these sites; and how to provide functional contexts to these sites. Obvious prioritizing criteria include good MS identification,

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into a consensus modification pattern.

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regulation of the site in a process of interest, a reasonable stoichiometry and/or localization of the site

Glycosylation of NaV1.5

Many ion channels are known to harbor glycan moieties which face the extracellular side of the membrane and anchor to residues such as serine, threonine (O-linked glycosylation) or asparagine (Nlinked glycosylation) [27] (Figure 2). These glycans are typically terminated by sialic acids which have been suggested to modulate voltage-gated ion channel function through their negative charges [28]. Glycosylation of the rat cardiac Na+ channel protein had been first demonstrated by Cohen and Levitt by using de-glycosidase enzymes [29]. It was shown that rat NaV1.5 has only about 13 kDa of carbohydrate, which is less than other neuronal isoforms with 50-60 kDa. More recently, Arakel and collaborators observed that the glycosylation pattern of NaV1.5 in mouse atrial tissues differs from ventricular tissues [30]. The migration patterns on immunoblots suggest that atrial NaV1.5 is more glycosylated than the proteins expressed in ventricular tissues, which may reflect chamber-specific maturation pathways. Despite the fact that one can predict more than five possible N-linked 8

ACCEPTED MANUSCRIPT glycosylation sites on extracellular asparagines of NaV1.5 (using the in silico NetNGlyc 1.0 algorithm [31]), thus far, there are no published studies that address the question of the residues which are actually modified in NaV1.5. Additionally, the great variety of glycan chains and the complexity of

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linkage to proteins make glycoproteomics a complicated application which relies on good glycoprotein

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or glycopeptide enrichment methods, intensive manual assignment of fragmentation spectra and still-

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maturing and growing glycan databases [32].

The Bennett group contributed substantially to understanding the role of sialylation in the

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regulation of NaV channel function. They first demonstrated that NaV1.5 is less glycosylated than the

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skeletal muscle NaV1.4 channel isoform in Chinese Hamster Ovary (CHO) cell lines, and that, when transiently transfected by itself, does not show any changes in voltage-dependent gating upon

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sialylation [33]. Nevertheless, in a subsequent study, they proposed a model in which the sialylation

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sensitivity of the cardiac NaV1.5 channel isoform is not mediated by the channel  subunit, but rather

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by the sialic acids that are N-linked to the associated/regulatory protein NaV1, and that all of the observed effects of NaV1 on channel  subunit gating depend on sialylation [14]. More specifically, the authors showed that the sialic acids linked to the extracellular N-terminal asparagines-93, -110, 114 and -135 of NaV1 (NCBI Reference Sequence NP_001028, Figure 1A), which conform to the Nglycosylation consensus sequence [N]X(S/T), are responsible for shifting the voltage-dependence of inactivation and activation towards hyperpolarized potentials, accelerating fast inactivation and slowing recovery from fast inactivation (Table 2). These effects of sialic acids have been assigned to an external negative surface potential which supposedly causes channels to gate following smaller depolarizations [14, 33]. The sialylation of cardiac NaV channels has been shown to be responsible for much of the hyperpolarizing shifts in NaV channel gating, as well as for the acceleration of fast inactivation and slowing of recovery from inactivation, that occur throughout the (neonatal to adult) development of the ventricle and between cardiac chambers (greater levels of functional sialic acids in 9

ACCEPTED MANUSCRIPT neonatal atria than ventricles) [34]. Nonetheless, the respective roles of the  or 1 channel subunits in these regulations could not be determined. The pathophysiological contribution of NaV1.5 glycosylation has been demonstrated in a study

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investigating the arrhythmogenic mechanisms in mice (MLP-/-) that are deficient for the Muscle LIM

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Protein which present with a heart failure phenotype [35] (Table 2). INa density in MLP-/- cardiac cells

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is smaller, the voltage-dependencies of inactivation and activation are altered, and rates of INa inactivation are slower. Immunoblot experiments suggested that the NaV1.5 channels in these MLP-/-

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tissues have an altered glycosylation pattern. Interestingly, similar Na+ current alterations were

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obtained when treating control cardiomyocytes with a specific de-glycosidase (neuraminidase). The pathophysiological role of reduced NaV1.5 sialylation has been further evidenced in mice (ST3Gal4-/-)

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deficient for the beta-galactoside alpha-2,3-sialyltransferase 4 (ST3Gal4) in which a rightward shift in

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ventricular NaV1.5 channel gating, slowing of fast channel inactivation and faster recovery from

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inactivation are associated with a shorter myocyte refractory period, a slower time to action potential peak and an increased susceptibility to ventricular arrhythmias [36] (Table 2). These two studies together demonstrate that one may pay attention to alterations in NaV channel glycosylation when investigating causes of cardiac disorders. Thus, it seems clear that the mechanisms and roles of glycosylation of the NaV1.5 channel as well as of the NaV1 associated/regulatory protein in physiology and disease states need to be further investigated in the future.

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Phosphorylation of NaV1.5 Phosphorylation is certainly the best documented PTM of cardiac NaV1.5 channels, with the

implication of various protein kinases recognized to affect diverse aspects of channel function. We review here the roles and molecular mechanisms of NaV1.5 channel regulation by the activation (or inhibition) of the kinase pathways that are known to affect the expression and/or the function of cardiac 10

ACCEPTED MANUSCRIPT NaV1.5 channels in normal and disease states. These pathways include Protein Kinases A (PKA) and C (PKC), Ca2+/Calmodulin-dependent protein Kinase II (CaMKII), Phosphatidylinositol 3-Kinase (PI3K), Serum- and Glucocorticoid-inducible Kinases (SGK), Fyn and Adenosine Monophosphate-

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activated Protein Kinase (AMPK) (Table 1). Although presented separately, it should be noted that

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potential physical promiscuity and associated functional overlap or interplay of different kinases that

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target a same phosphorylation site could exist. It is also expected, based on available literature on the phosphorylation of other ion channels in other systems, including neuronal NaV channels [37-39], that

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this current list of regulatory kinases will continue to grow in the next few years. While numerous in

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silico/in vitro studies of NaV1.5 channels have been effective in identifying a significant number of phosphorylation sites (nine in total, see Figure 1A), recent biochemical and proteomic advances have

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extended this list with the identification of seven additional native and previously unrecognized sites

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[18] (Figure 1B), thus bringing the total number to sixteen known sites. In general, commonly-used

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phosphorylation site prediction algorithms, ranging from simple consensus patterns to more advanced machine-learning algorithms, did not reliably predict in situ- and MS-identified sites [17, 18, 40, 41]. Application of these methods also clearly revealed that the number of phosphorylation sites that were identified, not only in NaV1.5, but also in various other membrane proteins, continues and will certainly continue to grow with the increasing sensitivity of proteomic instrumentation and methodologies. Whether these cutting-edge MS-based methods constitute the approach of choice for identifying in situ and physiologically relevant phosphorylation sites will be the goal of future electrophysiological investigations.

4.1.

PKA-dependent phosphorylation Although it is well-established that the cAMP-dependent Protein Kinase (PKA) pathway

regulates cardiac NaV1.5 channels, various effects have been reported in different cardiac cell preparations, and the significance of this regulation in cardiac physiology and/or pathophysiology has 11

ACCEPTED MANUSCRIPT not been clearly elucidated. -Adrenergic receptor activation modulates cardiac INa through both direct and indirect G-protein pathways [42, 43]. The indirect PKA-dependent phosphorylation pathway, of

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specific interest here, has been shown to cause either an increase [42, 44-50] or a decrease [51] in INa densities. Interestingly, however, a study using cell-attached macropatch in canine, rabbit, and guinea

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pig cardiomyocytes reported shifts in the voltage-dependence of both current inactivation and

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activation towards hyperpolarized potentials, whereas neither maximum conductance nor singlechannel conductance were changed [52]. The authors suggested that many of the seemingly disparate

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findings that were previously reported could be attributed to these observed shifts, implying the holding

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and test potentials that were used in the different experiments as the cause of discrepancies. Accordingly, the most reproducible finding in the various studies that were performed in

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cardiomyocytes was a negative shift of channel availability upon PKA activation [43, 45, 52-55]

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(Table 1).

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Using in vitro kinase assays on portions of the NaV1.5 protein, coupled with predictions that were based upon the consensus sequence for PKA-dependent phosphorylation (R/K)1-2(X)1-2[S/T], two phosphorylation sites in the first intracellular linker loop of the rat NaV1.5 protein have been attributed to PKA at serines-526 and -529 (S526 and S529, corresponding to S525 and S528 in the orthologous human cardiac NaV1.5 isoform, NCBI Reference Sequence NP_932173) [56] (Figure 1A). Subsequent studies in heterologous expression systems [44, 46, 47, 49, 50], and also cardiomyocytes [44, 45] showed, using a negative holding potential at which all channels are available, that PKA phosphorylation, in addition to regulating channel gating, also promotes trafficking of NaV1.5 channels to the cell plasma membrane (Table 1). It was observed that this effect is abrogated by mutation of the S525 and/or S528 phosphorylation sites, as well as a nearby putative endoplasmic reticulum retention signal (RRR) at position 533-535.

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ACCEPTED MANUSCRIPT The involvement of these PKA-dependent phosphorylation sites in cardiac disease has only been suggested in the context of two SCN5A genetic defects. The first one showed that the LQTS type 3 D1790G mutation results in PKA-dependent increase in late Na+ current through phosphorylation of

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both S36 and S525 [57] (Table 1). The second one demonstrated that the BrS-associated R526H

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mutation, located in the S528 PKA consensus phosphorylation site, results in both reduced basal Na+

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current densities, due to absence of phosphorylation and reduced cell surface channel expression, and absence of augmentation of current densities by -adrenergic stimulation [44] (Table 1).

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Although determination of the exact site(s) of phosphorylation could not be made,

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phosphoproteomic analyses of native NaV1.5 channels that were purified from adult mouse ventricles confirmed the existence of phosphorylation on S525, as well as on S36, in basal conditions [18]

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(Figure 1B). No phosphorylation was observed on S528. Note that these phosphoproteomic

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identifications were performed from ventricles at baseline, without any specific stimulation, and that

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the absence of detection of pS528 could be due to the lack of relevant (PKA) stimulation. Additionally, PKA-dependent phosphorylation of cardiac ion channels is usually mediated by adaptor proteins of the AKAP (A-Kinase Anchoring Protein) family that directly interact with the channel protein [58]. However, thus far, no specific cardiac AKAP has been reported to interact with NaV1.5.

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CaMKII-dependent phosphorylation Similar to regulation by PKA, the effects of Ca2+/Calmodulin-dependent protein Kinase II

(CaMKII) on cardiac Na+ currents have been debated. A first report in Human Embryonic Kidney cells 293 (HEK293) by Deschênes and collaborators suggested that Ca2+/Calmodulin-dependent protein Kinases (CaMK) modify NaV1.5 channel inactivation, although definitive conclusions could not be made because of different effects observed in response to two distinct CaMK inhibitors, KN93 and AIP [59]. A seminal work by the Maier group subsequently demonstrated a pivotal role for CaMKIIc, the 13

ACCEPTED MANUSCRIPT predominant cardiac cytosolic CaMKII isoform, in regulating NaV1.5 channel inactivation gating, both acutely in non-diseased rabbit ventricular myocytes and chronically in failing ventricular myocytes that were isolated from mice (CaMKIIc-Tg) overexpressing CaMKIIc [60]. Both acute and chronic

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overexpression of CaMKIIc shifts steady-state inactivation of Na+ channels to more negative

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potentials, enhances intermediate inactivation and slows recovery from inactivation in a Ca2+-

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dependent and supposedly interrelated manner (Table 1). Acute CaMKIIc overexpression also markedly slows fast INa inactivation and increases the density of INaL (Table 1). Consistently, several

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other studies in cells isolated from normal [61-64] or failing [65] ventricles found that Ca2+, calmodulin

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and/or CaMKII signaling slows inactivation of Na+ current and/or increases INaL. As suggested in the failing CaMKIIc-Tg mouse hearts [60] as well as in a pressure overload-induced heart disease mouse

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model [65], and also by computer simulations [66, 67], such alterations in Na+ channel function in the

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setting of increased CaMKII expression and activity may participate in action potential prolongation at

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slow heart rates and cardiac conduction slowing at shorter diastolic intervals, hence contributing to arrhythmogenesis. It is important to recognize, however, that other studies have shown different effects of CaMKII on Na+ currents [59, 68] and that caution must be taken, especially when interpreting findings obtained with different CaMKII isoforms, experimental designs and/or cell types. Characterization of the molecular mechanisms involved in the regulation of cardiac NaV1.5 channels by CaMKII is controversial. Using in silico predictions (based upon the putative consensus CaMKII phosphorylation site RXX[S/T]), the Mohler group first identified serine-571, in the first intracellular linker loop of NaV1.5, as a CaMKII phosphorylation site [69], whereas the Bers group later identified, using in vitro kinase assays coupled with in silico predictions, two other sites at positions S516 and T594 [70] (Figure 1A, Table 1). Consistent with previous analyses that showed that CaMKIIc co-immunoprecipitates with and phosphorylates endogenous NaV1.5 [60], the latter group added that CaMKIIc interacts with the first intracellular loop of NaV1.5 [70]. Additional studies 14

ACCEPTED MANUSCRIPT further showed increased phosphorylation at S571 (in association with the well-recognized increase in CaMKII expression and/or activity [71-73]) in common forms of heart disease [65], including human failing myocardium [74], thus supporting the role of CaMKII-dependent phosphorylation of S571 as a

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key player in dysregulating NaV1.5 channel function in heart failure (Table 1). The Mohler group also

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suggested the possibility that the two A572D and Q573E long QT3 syndrome variants in SCN5A,

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which neighbor S571, confer arrhythmia susceptibility by structurally and functionally mimicking CaMKII phosphorylation [74] (Table 1).

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While native NaV1.5 phosphoproteomic analyses confirm phosphorylation of S571 at baseline

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in mouse ventricles, no phosphorylation was observed at the other two suggested positions [18] (Figure 1B). It is also unclear whether: 1) this documented CaMKII-dependent regulation is

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intrinsically linked to the pre-association with and regulation by NaV1.5-bound calmodulin; 2) the

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effects on channel availability (inactivation from closed state) and late Na+ current (inactivation from

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open state) are mechanistically interrelated; and 3) the regulatory mechanisms involved in disease, compared to regulation at baseline, are similar and graded upon the increase in CaMKII expression and activity. Future investigations are certainly warranted to help develop a clearer mechanistic picture of how CaMKII regulates NaV1.5 channels in both normal hearts and during cardiac arrhythmias.

4.3.

PKC-dependent phosphorylation Initial studies in cardiac ventricular myocytes indicated that activation of Protein Kinase C

(PKC) reduces peak Na+ currents and shifts steady-state current inactivation in the hyperpolarized direction (Table 1), and that the observed reduced current could be attributed solely to a decreased probability of channel opening [75, 76]. Subsequent analyses in heterologous cells suggested that the effects on current density: 1) could largely be attributed to activation of the conventional, Ca2+sensitive PKC isoforms [77, 78]; 2) depend on, as does the shift in voltage-dependence of inactivation, phosphorylation of the previously recognized [79] and highly conserved serine-1503 (NCBI Reference 15

ACCEPTED MANUSCRIPT Sequence NP_932173) in the third intracellular linker loop of the channel [77, 80, 81] (Figure 1A); 3) require increased reactive oxygen species (ROS) [77]; and 4) conversely to initial reports [75], involve reduced channel trafficking to the cell surface [77, 78] (Table 1). Although no effects on channel

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inactivation kinetics were observed in these previous reports [75, 81], other studies demonstrated that

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activation of PKC, either directly or in response to application of increased intracellular Ca 2+

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concentration or hydrogen peroxide (H2O2), slows channel inactivation and/or increases INaL in ventricular myocytes [63, 82, 83] (Table 1). Together with the CaMKII findings, these data suggest

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that inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial

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dysfunction and cardiac arrhythmias that are caused by Ca2+ overload and/or enhanced INaL. Investigation of the physiological and pathophysiological consequences of PKC-mediated

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phosphorylation of cardiac NaV1.5 channels remained scarce until two mutations in the GPD1L gene,

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encoding the Glycerol 3-Phosphate Dehydrogenase 1-Like protein, were discovered in the Brugada

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(BrS) and the Sudden Infant Death (SIDS) syndromes. A first study in HEK293 cells reported that identified mutations in GPD1L decrease NaV1.5 channel cell surface expression and INa densities through PKC-dependent phosphorylation of S1503 [84] (Table 1). A parallel study extended this demonstration by showing that the increased intracellular NADH levels, resulting from or independent of the identified GPD1L mutations, decrease INa density through PKC activation and increased superoxide (O2-) in both HEK293 cells and cardiomyocytes, and increase the risk of ventricular tachycardia [85]. This latter study, however, demonstrated that this PKC-dependent regulation does not involve changes in cell surface channel expression. In addition, they did not investigate the role of direct phosphorylation by PKC. Importantly, these findings were confirmed in a mouse model of nonischemic cardiomyopathy in which elevated NADH levels, PKC activation, mitochondrial ROS overproduction and a concomitant decrease in INa were observed [86] (Table 1). Although uncertainties remain concerning the involvement of direct phosphorylation by PKC, these findings suggest that the 16

ACCEPTED MANUSCRIPT regulation of NaV1.5 channels by GPD1L, NADH and/or PKC may link the metabolic state of cardiomyocytes, particularly the reduction/oxidation (redox) and acidosis states, to membrane excitability and arrhythmia susceptibility.

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On the phosphoproteomic side, the presence of numerous tryptic cleavage sites (lysines)

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surrounding S1503 precluded detection of peptides and phosphopeptides comprising S1503 [18]

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(Figure 1B). Further evidence for native phosphorylation at S1503 and functional relevance of

PI3K- and SGK-dependent phosphorylation

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4.4.

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phosphorylation at this site in cardiomyocytes are therefore warranted.

Recent evidence from the group of Richard Lin reported that the prolongation of action

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potential duration and QT interval, in the context of drug-induced long QT syndrome [87] and diabetes

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[88], is mediated, at least in part, by inhibition of Phosphatidylinositol 3-Kinase (PI3K) and the

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subsequent increase in late Na+ current. Accordingly, mouse hearts lacking the PI3K p110 catalytic subunit exhibit prolonged action potential and QT interval that are at least partly a result of an increased INaL [87]. The authors further demonstrated that the PI3K-dependent increase in INaL in diabetic hearts is mediated through the inhibition of Protein Kinase B (PKB/Akt), a downstream effector of PI3K, and that inhibition of PKB/Akt by itself can also increase INaL in non-diabetic cardiomyocytes [88]. Together, these findings suggest that inhibition of the PI3K p110 subunit and downstream PKB/Akt mediates a common mechanism that adversely increases INaL density (Table 1) and subsequent risk of developing life-threatening arrhythmias. The Serum- and Glucocorticoid-inducible Kinases (SGK) are members of the serine/threonine protein kinase family that are acutely regulated both transcriptionally by several distinct signaling pathways, including the insulin or the IGF1 pathways, and post-translationally (phosphorylation/dephosphorylation), such as by receptor-activated PI3K [89]. Initial studies in 17

ACCEPTED MANUSCRIPT Xenopus oocytes demonstrated that SGK1 and SGK3, the two cardiac SGK isoforms, both increase Na+ current density, and that SGK3 further shifts voltage-dependence of inactivation and activation towards positive and negative potentials, respectively [90]. Opposite shifts in gating properties were elicited by

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individual mutations of the SGK consensus serines-484 or -664 (Figure 1A), suggesting direct

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involvement of phosphorylation at these two serines.

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Interestingly, the activity of SGK1 is increased in human and murine heart failure, and cardiacspecific expression of a constitutively active (S433D) SGK1 mutant in transgenic mice leads to cardiac

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dysfunction and ventricular arrhythmias [91]. Cardiomyocytes from transgenic mice showed a

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hyperpolarizing shift in the voltage-dependence of Na+ current inactivation and activation, in addition to increased Na+ current density in comparison with WT cardiomyocytes (Table 1). One of the

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functions of SGK1 is to phosphorylate and inactivate the ubiquitin ligase Nedd4-2 [92], which in turn

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leads to decreased binding of Nedd4-2 to NaV1.5 and increased NaV1.5 cell surface expression. This

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mechanism has been suggested to mediate the increased INa in the S433D-SGK1 cardiomyocytes [91]. Additionally, the density of INaL is increased in S433D-SGK1 cardiomyocytes (Table 1) and proarrhythmic effects of SGK1 could be reversed by selective block of INaL with ranolazine [91]. Conversely, cardiac-specific expression of a dominant negative (K127M) SGK1 mutant protects mice after hemodynamic stress from adverse cardiac dysfunction and Na + channel alterations [91]. Although PKB/Akt and SGK1 are both downstream effectors of PI3K and display structural similarities, the effects of PI3K-PKB/Akt and SGK1 on the late Na+ current (although described in two distinct pathological contexts) seem opposite, and it remains unclear whether the three kinases (PI3K, PKB/Akt, and SGK) act synergistically or separately and whether the underlying regulatory mechanisms involve (common) changes in the phosphorylation status of NaV1.5. While endogenous cardiac SGK1 co-immunoprecipitates with NaV1.5 [91], such information is not available for PI3K or PKB/Akt. Intriguingly, the two predicted SGK1 phosphorylation sites at positions S484 and S664 [90] (NCBI Reference Sequence NP_932173), which have been detected in NaV1.5 proteins that were 18

ACCEPTED MANUSCRIPT purified from native cardiac tissues [18] (Figure 1B), both conform to the preferred and common phosphorylation target sequence for SGK1 and PKB/Akt, RXRXX[S/T]. Further determination of the functional contribution of these or other phosphorylation sites in the PI3K-PKB/Akt- and/or SGK1-

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dependent regulation of cardiac NaV1.5 channels (in particular, in the generation of the late Na+ current

4.5.

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in heart failure, diabetes, and drug-induced LQT syndrome) is therefore warranted.

Fyn-dependent phosphorylation

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NaV1.5 is also the target of the Src-family tyrosine kinase Fyn [93]. The effects of Fyn on

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heterologously-expressed NaV1.5 channels are manifested as a depolarizing shift of steady-state current inactivation, an increased rate of recovery from inactivation, and a decreased entry rate into

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intermediate inactivation (Table 1). In vitro biochemical and functional evidence suggested that this

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effect depends on the phosphorylation of tyrosine Y1495, seven amino acids downstream of the IFM

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inactivation "ball" in the third intracellular linker loop of the channel (Figure 1A), and that cardiac NaV1.5 channels are indeed tyrosine-phosphorylated. Consistently, another study reported that the protein tyrosine phosphatase, PTPH1, which interacts with the C-terminal PDZ domain of NaV1.5, inversely shifts the voltage-dependence of inactivation towards hyperpolarized potentials [94]. Interestingly, opposite results were obtained with the neuronal NaV1.2 channels for which an increased rate of inactivation and a hyperpolarizing shift in the voltage-dependence of inactivation were observed upon Fyn co-expression [95, 96]. Altogether, these results suggest that phosphorylation/dephosphorylation of Y1495 [93] and other [96] tyrosine residue(s) contribute directly to modulating the stability of inactivation in one way or the other, depending on NaV channel subtypes. Phosphorylation of Y1495 from purified cardiac NaV1.5 channels was not detected using mass spectrometry [18] (Figure 1B), possibly due to the distinctly low levels of tyrosine phosphorylation compared with phosphoserines and phosphothreonines [97].

19

ACCEPTED MANUSCRIPT 4.6.

AMPK-dependent phosphorylation Cardiac NaV1.5 channels may also be substrates for the Adenosine Monophosphate-activated

Protein Kinase (APMK), a regulatory mechanism that has been suggested to be responsible for the

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arrhythmogenic activity observed in Wolff-Parkinson-White patients that present with AMPK

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mutations and associated increased AMPK activity [98] (Table 1). In this study, the authors showed, in

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a mammalian cell line, that a constitutively active AMPK mutant slows NaV1.5 channel inactivation with the appearance of a persistent Na+ current, and shifts the voltage-dependence of NaV1.5 channel

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activation towards hyperpolarized potentials (Table 1). These findings were corroborated in

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adenoviral-infected cultured rat ventricular myocytes which demonstrated prolonged action potential duration and early afterdepolarizations. Localization of involved phosphorylation sites was not

Arginine methylation and N-terminal acetylation of NaV1.5

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5.

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investigated.

Four recent studies from the group of Ramon Brugada suggested the role of arginine methylation and alanine acetylation (Figure 2) in the modulation of cardiac NaV1.5 channels. A first proteomic analysis from a stable cell line that expresses NaV1.5 provided the first evidence that the arginines R513, R526, and R680, located in the first intracellular linker loop of NaV1.5, are modified by methylation [15] (Figure 1A). Each of the three arginines was found to be monomethylated; R526 and R680 were also detected in a dimethylated state. The functional relevance of these findings was underscored by the fact that R526H and R680H are mutations known to cause Brugada and long QT type 3 syndromes, respectively (Table 2). A second study further demonstrated that the protein arginine methyl transferases (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in HEK293 cells, and increase NaV1.5 cell surface expression and Na+ current density [99] (Table 2). Finally, these findings have recently been supported by the mass spectrometric identification of monoand dimethylation of R526 from native NaV1.5 channels that were purified from end-stage failing 20

ACCEPTED MANUSCRIPT human ventricles (Figure 1B, Table 2) [16]. In these native samples, methylated R513 and R680 were not detected. Even more intriguing is the existence of an N-terminus devoid of the initiation methionine and acetylated at the resulting initial alanine (AcA2) residue in human cardiac NaV1.5 protein purified

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from failing hearts [16] (Figure 1B, Table 2). Future studies, aimed at investigating the roles of these

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novel processing in the regulation of cardiac NaV1.5 channels, both at baseline and in heart failure, are

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undoubtedly of great interest. Of particular interest is the reciprocal regulation of phosphorylation (decreased phosphorylation) and methylation (increased methylation, including at R513) at adjacent

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sites, which was recently suggested to underlie the changes in neuronal NaV1.2 channel function in

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response to acute seizures [100]. In this respect, one can speculate that arginine methylation may have a role in regulating phosphorylation of key serine residues, or vice versa, especially in the first

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intracellular linker loop of NaV1.5 channels which is highly prolific for both methylation and

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phosphorylation. This antagonistic interplay was recently demonstrated for NaV1.5: in vitro R513

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methylation decreases S516 phosphorylation, and reciprocally, S516 phosphorylation blocks R513 methylation [101]. The authors of this recent study also suggested that the G514C mutation in NaV1.5, associated with cardiac conduction disease, could act by balancing this methylation/phosphorylation equilibrium (Table 2).

6.

Redox regulation of NaV1.5 The reduction/oxidation (redox) sensitivity of cardiac NaV1.5 channels involves several distinct

mechanisms: regulation of the SCN5A promoter [102, 103], regulation of channels by redox-activated proteins/pathways (particularly kinases), and direct chemical modification of channel subunits by reactive oxygen species (ROS). Here, we focus on only those two latter mechanisms involving PTMs of NaV1.5. ROS are comprised of superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH) and peroxynitrite (ONOO-). They are generated by several metabolic pathways, which involve 21

ACCEPTED MANUSCRIPT uncoupled nitric oxide synthases (NOS), the NAD(P)H oxidases, xanthine oxidases, and the mitochondrial electron transport chain (ETC), which is the major cardiac ROS source. Although of direct relevance in pathological conditions such as heart failure or ischemia, where ROS are elevated

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[104], it is important to stress that these ROS-dependent signaling pathways and regulations may not

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necessarily be detrimental and may also be involved in physiological processes.

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As discussed above (in part 4.3), increased intracellular NADH, whether in the setting of BrSor SIDS-associated GPD1L mutations or in the context of non-ischemic cardiomyopathy, reduces

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cardiac INa (Table 2). This implies, in addition to a potential PKC-dependent phosphorylation of

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NaV1.5 [84], an increased production of superoxide [85, 86]. The Dudley group further extended these findings by showing that the main source of production of superoxide that is induced by elevated

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NADH in this context is the complex III of mitochondrial ETC [105]. No unequivocal underlying

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mechanisms implying direct redox modifications of the NaV1.5 protein have been established.

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Several evidence also demonstrated that ROS enhance late INa, in particular in the context of heart failure (Table 2). Although a first study in HEK293 cells suggested the direct involvement of methionine oxidation (Figure 2) at multiple conserved NaV  subunit residues, including methionine1487 in the IFM inactivation "ball" [106], the group of Lars Maier later observed that the acute H2O2dependent increase in late INa is absent in CaMKII-deficient myocytes [107], thus demonstrating that the dominating mechanism involved in this regulation is not mediated by direct NaV1.5 thiol oxidation, but rather depends on the indirect oxidation and subsequent activation of CaMKIIc, and possibly phosphorylation of NaV1.5. From a technological point of view, it should be noted that the proteomic identification of methionione oxidation has been hampered by experimental limitations due to the fact that this modification occurs when proteins are exposed to air during sample preparation, and that special techniques need to be applied to circumvent this issue [108].

22

ACCEPTED MANUSCRIPT The third source of ROS that is of relevance to the regulation of NaV1.5 channels is nitric oxide (NO) which, in the cardiovascular system, is mainly synthesized by endothelial NOS (eNOS) in the coronary endothelium. NO is also produced within the cardiac myocytes themselves by the

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constitutive neural NOS (nNOS). NO has been suggested to act on proteins, including NaV1.5, through

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at least two distinct pathways: via direct S-nitrosylation of sulfhydryl groups of specific cysteine

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residues (Figure 2), and through indirect activation of the guanylyl cyclase (GC)/cGMP pathway. A

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first study in 2000 reported that an increase in Ca2+ loading, induced by ionomycin, increases INaL in adult rat ventricular myocytes, and that this effect, which was blocked by inhibitors of NOS, but not by

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inhibitors of the GC/cGMP pathway, might depend on the direct chemical modification of NaV channels [109] (Table 2). In contrast, another study demonstrated that NO reduces INa in guinea pig

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and mouse ventricular myocytes (the channel conductance or gating were unchanged), and that this

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effect is mediated by both the GC/cGMP/Protein Kinase G (PKG) and the adenylyl cyclase

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(AC)/cAMP/PKA pathways [110]. Importantly, by studying the effects of carbon monoxide (CO) on both native and heterologously-expressed NaV1.5 channels, two additional studies somehow reconciled these observations by showing that CO, mostly through the formation of NO and modification of the redox status of the channels, was able to both increase INaL and decrease INa [111, 112]. The regulation of NaV1.5 channels by NO has further been explored in the context of two LQTS (LQT12) mutations identified in the cytoplasmic sub-membranous protein 1-syntrophin (SNTA1 gene, Table 2). 1-syntrophin interacts with and acts as a molecular scaffold for both nNOS and the cardiac plasma membrane Ca-ATPase, PMCA4b (an inhibitor of nNOS and NO synthesis [113]), as well as the NaV1.5 channels through its binding to the C-terminal PDZ binding domain [114]. The LQTS A257G-SNTA1 mutation was found to cause a leftward shift of the activation curve (increasing the window currents), an increased current density and a slowed fast inactivation [115]. The LQTS A390V-SNTA1 mutation was suggested to disrupt association with PMCA4b, therefore releasing 23

ACCEPTED MANUSCRIPT inhibition of nNOS, and causing direct S-nitrosylation of NaV1.5 and increased INaL [113]. nNOSdependent direct S-nitrosylation of NaV1.5 channels has also been suggested to mediate the increased INaL observed in the context of the LQT9-associated mutation in caveolin-3, Cav3-F97C [116] (Table

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2). These findings together suggest that direct S-nitrosylation and consequent gain-of-function of

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NaV1.5 channels may account for the development of arrhythmias in LQTS patients.

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The impact of ROS on cardiac INa may also be mediated by direct lipoxidation of NaV1.5 channels, which covalently modifies lysine residues and forms isoketal stable adducts [117, 118].

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These chemical modifications have been suggested to occur on the NaV1.5 protein in the context of

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acute myocardial infarction, which is accompanied by extensive oxidative injury and is, in part, characterized by increased levels of isoketal protein adducts and reduced channel availability (Table

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2). These channel defects that are associated with disease have been validated, both in heterologous

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cells and in the cardiac-derived HL-1 myocyte cell line, by exogenous addition of highly reactive -

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ketoaldehydes that were generated by the peroxidation of arachidonic acid [118]. In addition, these effects were prevented by pretreatment with selective isoketal scavengers. To date, however, specific sites for reactive lipoxidation on NaV1.5 or its associated/regulatory proteins remain to be determined.

7.

Ubiquitylation of NaV1.5

The cardiac NaV1.5 channel was the first ion channel that was shown to be ubiquitylated in cardiac tissues [119]. Ubiquitylation of membrane proteins is known to be one of the important cellular mechanisms that is involved in their internalization process [120]. Ubiquitylation of NaV1.5 and other members of the NaV channel subfamily [121] is achieved by interaction with the E3 ubiquitin ligase Nedd4-2, a member of the Nedd4 family (neuronal precursor cell developmentally down-regulated 4) of E3 ubiquitin ligases [122]. Van Bemmelen and collaborators demonstrated that the ubiquitylation of NaV1.5 depends on the catalytic activity of Nedd4-2 by comparing wild-type with a catalytically dead 24

ACCEPTED MANUSCRIPT mutant of Nedd4-2, Nedd4-2-C867S, in HEK293 cells [119]. This PTM occurs via the interaction between the PY-motif (X(P/L)PXY) at the C-terminus of NaV1.5 and the fourth tryptophan rich domain (WW) of Nedd4-2 [119, 123], as previously described with the epithelial Na+ channel ENaC [124].

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Note that this Nedd4-2 interacting PY-motif is also found in other isoforms of NaV channels [121],

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cardiac K+ channels such as KCNQ1 [125] and hERG [126], and connexin 43 [127]. By using brefeldin

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A, a compound known to block the forward trafficking of newly synthesized proteins from the transGolgi network towards the plasma membrane, it was shown that co-expression of Nedd4-2 increases

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the rate of internalization of NaV1.5 channels [123] (Table 2). In addition, it has been demonstrated

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that NaV1.5 is also ubiquitylated in the murine heart [119], suggesting that this PTM is involved in the regulation of NaV1.5 channels in vivo. In a study by Kang and collaborators, a specific up-regulation of

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NaV1.5 channels and INa was observed in neonatal rat cardiomyocytes that were treated for 24 hours

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with bepridil, an anti-arrhythmic agent [128]. This effect was dependent on the activity of the

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proteasome, thus suggesting once again, the role of the ubiquitin-proteasome pathway in regulating the cell surface expression of NaV1.5 channels in cardiac cells. More recently, chronic inhibition of the proteasome by treating mice for 7 days with the proteasome inhibitor MG132 rescued the amount of NaV1.5 protein and INa that was decreased in dystrophin-deficient (mdx) mice [129]. While these aforementioned studies demonstrate that NaV1.5 is a target of Nedd4-2-dependent ubiquitylation and that this PTM is involved in the control of the number of channel proteins expressed at the cell surface, we are still lacking a detailed understanding about the mechanisms involved. It is not clear which lysine (or other) residues are ubiquitylated in vivo, and we do not have any information about the type of ubiquitin chains that are tagging NaV1.5 for internalization. The MS identification of ubiquitylation sites has proven to be difficult because of the low stoichiometry of ubiquitylated proteins and the large size and diversity of ubiquitin chains. The use of antibodies specific for the di-glycyl remnant, which are produced on ubiquitylated lysines (K-GG) after trypsin digestion, should nevertheless improve our ability to enrich and detect endogenous ubiquitylation sites by MS [130]. 25

ACCEPTED MANUSCRIPT Furthermore, a few recent studies have implicated de-ubiquitylating enzymes of the USP family, in particular USP2, in counteracting the effects of Nedd4-2 on ion channel proteins [131, 132]. No such de-ubiquitylating enzymes have so far been described for NaV1.5 or other members of the NaV channel

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subfamily. Lastly, under certain pathological conditions such as nerve injury that leads to neuropathic

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pain, the Nedd4-2 level of expression has been shown to be reduced in mouse sensory neurons [133]. It

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may be worthwhile to look for cardiac pathological conditions in which similar down-regulation of

Conclusions and perspectives

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8.

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Nedd4-2 expression may lead to a hyper-excitable phenotype.

In this review article, we summarized for the first time the current knowledge demonstrating

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that many amino acid residues of the NaV1.5 (and NaV1) channel proteins can be post-translationally-

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modified. Some of these PTMs have been proposed to play critical roles in regulating the expression

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and the function of this key cardiac ion channel. We have described about half a dozen of such modifications. However, this is most likely only the tip of the iceberg since the current list of possible PTMs exceeds 400 [134]. Among the possible additional modifications that have been reported for ion channel proteins, one can mention sumoylation [120], which is similar to ubiquitylation but achieves other functions, and palmitoylation [135] which is most of the time, a signal to anchor proteins into membranes. However the possibilities of PTMs on a protein with more than 2000 amino acids such as NaV1.5 are large! Clearly, it is difficult to predict how many PTMs there are and the extent of their complexity, as well as their functions under physiological conditions and in disease states. Technological progress in the field of mass spectrometry and proteomics are rapid [136], and it is conceivable that, with greater mass spectrometry capabilities, many additional and/or lower abundant PTM-bearing peptides could be detected without prior and specific enrichment. This would make it possible to study different PTMs simultaneously and to directly estimate their relative abundance as 26

ACCEPTED MANUSCRIPT well as stoichiometry from biological samples under various circumstances. We can therefore expect to learn a lot in the coming years about how not-yet-described PTMs of NaV1.5 may explain the still-to-be discovered functions of this channel and what their roles are in cardiac disorders, and in

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arrhythmogenesis in particular.

27

ACCEPTED MANUSCRIPT Highlights The cardiac NaV1.5 channel is subject to PTMs whose roles are partially understood.



The majority of NaV1.5 PTM sites are located in the first loop of the channel.



The use of native channel proteomics is of high discovery and gain.



NaV1.5 PTMs contribute to cardiac disease, which may have therapeutic applications.

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

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Acknowledgments We are grateful to Diana Shy for her comments on this manuscript, and acknowledge financial support provided by the Marie Curie 7th Framework Program of the European Commission (NavEx256397 to C.M.), the Fondation d’entreprise Genavie (to C.M.), the Swiss National Science Foundation (310030_14060 to H.A.) and the European Community's 7th Framework Program FP7/2007-2013 under grant agreement (no. HEALTH-F2-2009-241526, EUTrigTreat to H.A). We would also like to thank Josh Gramling for his contribution in the drawing of the figures.

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Disclosures None.

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ACCEPTED MANUSCRIPT 9.

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. Localization of post-translational modification sites on the human cardiac NaV1.5 and Nav1

PT

channel subunits. Findings obtained using in silico and/or in vitro analyses (A) and native proteomic analyses from cardiac tissues (B) are compared directly. From a total of twenty-four PTM sites, sixteen

RI

have been suggested by using in silico/in vitro analyses, and thirteen have been identified using native

SC

proteomics. Out of the sixteen sites identified in silico/in vitro, only five sites (pS484, pS525, meR526, pS571 and pS664) have been observed by MS. Proteomic analyses from cardiac tissues added a total of

NU

seven previously unrecognized phosphorylation (in red) and one novel N-terminal acetylation (in dark

MA

blue) sites, for which modifying enzymes (kinases and N-terminal acetyltransferases, respectively) remain to be characterized. When documented, the implication of specific kinases in phosphorylating

D

identified sites is color-coded. Three and two phosphorylation site locations are possible at amino acids

TE

36 to 42 and 524 to 525, respectively. With the exception of two residues located in the cytoplasmic N-

AC CE P

terminus (pS36, pT38 and/or pS42, and AcA2), and two residues in the third intracellular linker loop (pY1495 and pS1503), all the sites identified are clustered in the first intracellular linker loop, making it a hotspot for post-translational regulation. Note that PTM sites have originally been identified from the mouse, rat or human NaV1.5 sequences, and that the PTMs reported here are numbered on the conserved amino acid residues of the human NaV1.5 isoform a (NCBI Reference Sequence NP_932173). The human NaV1 NCBI Reference sequence used is NP_001028.

Figure 2. Chemical structures of post-translationally-modified amino acid residues. Amino acid backbones are represented in black and post-translational additions are highlighted in red. Abbreviation: Ub, ubiquitin.

35

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Table 1. Most consistent effects and suggested pathological implications of cardiac NaV1.5 channel phosphorylation

PI3K and PKB/Akt

SGK

Fyn

AMPK *

- Shifts voltage-dependence of inactivation towards negative potentials - Increases NaV1.5 trafficking and INa

S516, S571 and T594

S571 (mouse, dog, human)

- Shifts voltage-dependence of inactivation towards negative potentials - Enhances intermediate inactivation - Slows recovery from inactivation - Slows fast INa inactivation and increases INaL

S1503

No

Unknown

S484 and S664

RI

PT

Species

Involvement in inherited or acquired cardiac diseases

Associated SCN5A mutations and phosphosites

References

- LQT3 - BrS

- D1790G (pS36 and pS525) - R526H

42-50, 52-57

- Het. cells - mouse, guinea pig, rabbit, dog

- LQT3 - Heart failure

- A572D, Q573E - pS516, pS571 and pT594

59-65, 69, 70, 74

- Shifts voltage-dependence of inactivation towards negative potentials - Decreases NaV1.5 trafficking and INa - Slows fast INa inactivation and increases INaL

- Het. cells - Mouse, rat, guinea pig, rabbit

- BrS and SIDS - Heart failure

- GPD1L mutations - N/A

63, 75-78, 80-86

Unknown

Inhibition increases INaL

Mouse, dog

- Drug-induced LQT - Diabetes

N/A

87, 88

S484 and S664 (mouse)

- Phosphorylates and inactivates Nedd4-2, and increases NaV1.5 cell surface expression and INa - Shifts voltage-dependence of inactivation and activation towards negative potentials - Increases INaL

- Het. cells - Mouse

Heart failure

N/A

90, 91

- Shifts voltage-dependence of inactivation towards positive potentials - Accelerates recovery from inactivation - Decreases intermediate inactivation

Het. cells

N/A

N/A

93

- Shifts voltage-dependence of activation towards negative potentials - Slows fast INa inactivation and increases INaL

- Het. cells - Rat

Wolff-Parkinson-Whiteassociated cardiomyopathies

AMPK mutations

98

Y1495

No

Unknown

Unknown

NU

SC

- Het. cells - Rat, guinea pig, rabbit, dog

MA

PKC

S525 (mouse) and S528 (rat)

Most consistent effects on cardiac Na currents

PT ED

CaMKII

S525 and S528

+

CE

PKA

Validation in cardiac cells

AC

Kinase(s)

Phosphorylation * site(s)

Phosphorylation sites are numbered on the human NaV1.5 sequence (NCBI Reference Sequence NP_932173). Abbreviations: BrS, Brugada syndrome; GPD1L, Glycerol 3-Phosphate Dehydrogenase 1-Like; Het. cells, Heterologous cells; LQT, Long QT syndrome; SIDS, Sudden Infant Death Syndrome.

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Table 2. Most consistent effects and suggested pathological implications of post-translational modifications of cardiac NaV1.5 and NaV1 channel subunits Species

PT

- Shifts voltage-dependence of inactivation and activation towards negative potentials - Accelerates fast INa inactivation - Slows recovery from inactivation

+

Involvement in inherited or acquired cardiac diseases

Associated SCN5A mutations and PTMs

References

- Glycosylation-associated cardiomyopathies - Heart failure

N/A

13, 34-36

Het. cells

- Cardiac conduction disease - BrS - LQT3 - Heart failure

- G514C (meR513 and pS516) - R526H - R680H - meR526

15, 16, 99, 101

N/A

Heart failure

AcA2

16

RI

No

Most consistent effects on cardiac Na currents

- Het. cells - Mouse, rat

SC

N93, N110, N114 and N135 ** on NaV1

Validation in cardiac cells

R513, R526 and R680

R526 (human)

Increases NaV1.5 cell surface + expression and Na current density

N-terminal acetylation

A2

A2 (human)

Unknown

Regulation by NADH/O2

Unknown

Unknown

Reduces INa

- Het. cells - Mouse, rat

- BrS and SIDS - Heart failure

- GPD1L mutations - N/A

85, 86, 105

Regulation by H2O2

Unknown

Unknown

Oxidizes and activates CaMKIIc, and increases INaL

- Het. cells - Mouse, rabbit

Heart failure

N/A

106, 107

S-nitrosylation

Unknown

Unknown

Increases INaL

- Het. cells - Mouse, rat

- LQT9 - LQT12

- Cav3 mutation - SNTA1 mutations

109, 111, 112, 113, 115, 116

Lipoxidation

Unknown

Unknown

Shifts voltage-dependence of inactivation towards negative potentials

- Het. cells - HL-1 myocytes

Myocardial infarction

N/A

117, 118

Ubiquitylation

Unknown

Unknown

Increases NaV1.5 internalization and decreases INa

- Het. cells - Mouse, rat

N/A

N/A

119, 123, 128, 129

CE

PT ED

MA

Arginine methylation

AC

N-Glycosylation

Site(s) on * NaV1.5

NU

Post-translational modification

Sites of post-translational modifications are numbered on the human NaV1.5 and NaV1 sequences (NCBI Reference Sequences NP_932173 and NP_001028, respectively). Abbreviations: BrS, Brugada syndrome; Cav3, Caveolin-3; GPD1L, Glycerol 3-Phosphate Dehydrogenase 1-Like; Het. cells, Heterologous cells; HL-1 myocytes, Cultured mouse atrial myocyte cell line; LQT, Long QT syndrome; SIDS, Sudden Infant Death Syndrome; SNTA1, 1-syntrophin gene. *

**

2

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

2