Phosphorylation of cardiac voltage‐gated sodium channel: Potential players with multiple dimensions

Abstract Cardiomyocytes are highly coordinated cells with multiple proteins organized in micro domains. Minor changes or interference in subcellular proteins can cause major disturbances in physiology. The cardiac sodium channel (NaV1.5) is an important determinant of correct electrical activity in cardiomyocytes which are localized at intercalated discs, T‐tubules and lateral membranes in the form of a macromolecular complex with multiple interacting protein partners. The channel is tightly regulated by post‐translational modifications for smooth conduction and propagation of action potentials. Among regulatory mechanisms, phosphorylation is an enzymatic and reversible process which modulates NaV1.5 channel function by attaching phosphate groups to serine, threonine or tyrosine residues. Phosphorylation of NaV1.5 is implicated in both normal physiological and pathological processes and is carried out by multiple kinases. In this review, we discuss and summarize recent literature about the (a) structure of NaV1.5 channel, (b) formation and subcellular localization of NaV1.5 channel macromolecular complex, (c) post‐translational phosphorylation and regulation of NaV1.5 channel, and (d) how these phosphorylation events of NaV1.5 channel alter the biophysical properties and affect the channel during disease status. We expect, by reviewing these aspects will greatly improve our understanding of NaV1.5 channel biology, physiology and pathology, which will also provide an insight into the mechanism of arrythmogenesis at molecular level.

calmodulin-dependent kinase II (CaMKII) are among the most abundant kinases expressed in the left ventricle of the heart, according to proteomic studies. 4 Na V 1.5 channel function and its regulation are in themselves complex processes, becoming ever more complex as new interacting protein partners are identified. In this review, we summarize structure and function of the Na V 1.5 channel, formation of the macromolecular complex, its subcellular distribution and modulation by phosphorylation.

| STRUCTURE AND FUNCTION
The cardiac sodium channel consists of one α-(Na V 1.5) and one or more auxiliary β-subunits in a 1:1 ratio. The Na V 1.5 adult or canonical isoform is composed of 2016 amino acid residues with a molecular mass of about 260 kDa. [5][6][7] Five different β-subunits (β 1 -β 4 and β 1B ) are expressed in cardiac tissue. The β-subunits share a common membrane topology including an extracellular N-terminal that adopts an immunoglobulin fold, a transmembrane domain and an intracellular C-terminal domain. The subunit β 1B is an exception that is a splice variant of β 1 which lacks a transmembrane domain. The β 1 and β 3 -subunits associate with the Na V 1.5 channel α-subunit noncovalently, while β 2 and β 4 -subunits are linked covalently by disulfide bonds. 5,8 These non-pore forming β-subunits are implicated in the physiology and pathology of the α-subunit and play an important role in regulating the kinetics, gating, surface expression and voltage dependence of the Na V 1.5 channel. 5,9 Na V 1.5 α-subunit RNA is a product of 28 different exons. Exon 1 and part of exon 2 encode the 5′-untranslated region; the protein-coding region spans exons 2-28, while the 3′-untranslated region is encoded by exon 28. 1 Alternative splicing results in the production of several Na V 1.5 RNA transcripts which can be categorized into functional (Na V 1.5a, Na V 1.5c, Na V 1.5d, Na V 1.5e and hH1c) and non-functional (Na V 1.5b, Na V 1.5f and C-terminal splice variant) splice variants. 7,10 Na V 1.5 channel protein has a modular structure consisting of four domains (D I -D IV ), which are connected by intracellular connecting loops (ICL I-II , ICL II-III , and ICL III-IV ). In addition to intracellular connecting loops, both carboxyl terminus (C-terminus) and amino terminus (N-terminus) are also located intracellularly. Each domain is further comprised of six transmembrane segments (S 1 -S 6 ), which are connected by short, alternating, intra-and extracellular loops. 11 The transmembrane subunit S 4 of each domain contains positively charged amino acids at every third or fourth position and serves as a voltage sensor. 12 The S 5 and S 6 subunits of each domain constitute the pore lining, and are connected by loops called P-loops which curve back into the pore and form the selectivity filter (a group of four amino acid residues: aspartic acid, glutamic acid, lysine and alanine; DEKA arrangement). Of these four amino acids, lysine in D III is vital for differentiation between monovalent Na + and divalent Ca ++ ions ( Figure 1). [13][14][15] Voltage-dependent gating is a process by which alteration in membrane potential, results in structural conformations causing the ion channels to open (conductive) or close (nonconductive). Na V 1.5 channels are activated (opened) by the outward movement of voltage sensor S 4 and allow Na + influx. Initially, Na + permeability increases rapidly during phase 0 of the action potential and then decreases due to Na V 1.5 channel inactivation (closed), which renders Na V 1.5 refractory until repolarization is completed. 6 The kinetics of inactivation can be subdivided into slow inactivation which develops over several seconds and regulates excitability, while fast inactivation which occurs within milliseconds is important in action potential repolarization. 16,17 The process of slow inactivation is not well understood; involvement of P-loops and various conformational states are assumed to lead the channel into slow inactivation. 16,18 The mechanism of fast inactivation on the other hand, is well established and an inactivation gate comprising the amino acid residues isoleucine, phenylalanine and methionine (IFM), has been identified in ICL III-IV . Scanning mutation analysis has identified several amino acids in the short intracellular connecting loops of transmembrane segments S 4 and S 6 in D III and D IV , which serve as docking sites for inactivation and closing of the channel pore. 13,15 The C-terminus is also known to modulate Na V 1.5 channel inactivation by stabilizing and minimizing channel reopening. 19 Na V 1.5 channel activation derives its voltage dependence from outward movement of the voltage sensor, S 4 , in response to alteration in membrane potential. This outward movement of the S 4 subunit also initiates inactivation, thus deriving its voltage dependence by coupling with the process of activation. 6,20 Na V 1.5 channels can be activated again during phase 4 of the action potential after recovering from inactivation, although, some channels (< 1%) may reactivate during phase 2 and 3 of the action potential and generate small late sodium current (late I Na ), also called persistent noninactivating current. This small inward current is usually less than 0.5% of the peak I Na but it flows approximately 300-400 milliseconds longer thereby maintaining action potential plateau and playing an important role in Na + loading. Increased intracellular Na + levels also increase Ca ++ levels via the Na + /Ca ++ -exchanger hence also affecting contraction and relaxation. 21 Late I Na has minimal contribution to the action potential under physiological conditions, but plays an important role in the pathological context. Late I Na is increased in acquired disease conditions like heart failure (HF), hypertrophy and diabetes mellitus (DM) or under congenital cardiac disorders like long-QT syndromes (LQTS). 21,22 This increased Late I Na can trigger early afterdepolarizations by prolonging action potential duration or delayed afterdepolarizations by increasing intracellular Ca ++ levels thus contributing to arrythmogenesis. 21,23,24 Alterations in the SCN5A gene can lead to Na V 1.5 channel dysfunction resulting in either gain-of-function or lossof-function effects. These mutations in the Na V 1.5 channel affect structure, function, trafficking, interaction with other protein partners and formation of the macromolecular complex. Na V 1.5 channel variants have been associated with several congenital cardiac disorders such as atrial standstill, atrial fibrillation (AF), Burgada syndrome (BrS), cardiac conduction disease (CCD), dilated cardiomyopathy (DCM), LQTS and sudden infant death syndrome (SIDS). 25,26 Gain-of-function mutations in Na V 1.5 result in increased persistent current which may lead to long-QT syndrome type 3 (LQTS-3), while loss-of-function mutations result in decreased peak I Na and are implicated in BrS and sick sinus syndrome (SSD). The phenotype of these Na V 1.5 channel mutations depends on several factors which may include genetic, transcriptional, translational and post-translational modifiers. Discussion of these modifiers affecting the phenotype of disease-causing mutant channels is  (ICL I-II -ICL III-IV ). Each domain is further comprised of six transmembrane subunits (S 1 -S 6 ). The S 4 subunit of each domain constitutes the voltage sensor while IFM motif in ICL III-IV plays a critical role in channel inactivation. The extracellular loop between S 5 and S 6 of each domain form P-loop which acts as a selectivity filter. Various Na V 1.5 protein partners are shown in different colours which correspond to the formation of macromolecular complex as shown in Figure 2. The position of these protein partners in IC-loops, N-and C-terminal is according to their approximate interaction site with the Na V 1.5 α-subunit beyond the scope of this review and we refer interested readers to some excellent review articles for further reading. 9,26,27

AND SUBCELLULAR DISTRIBUTION
Cardiac myocytes are rod-shaped cells, approximately 100 μm in length and 20 μm in width, expressing myriad of proteins in different membrane compartments. These proteins are precisely localized indicating their distinct functional roles. Depending on subcellular localization, Na V 1.5 channels are known to be arranged in three different compartments on plasma membrane, namely intercalated discs, lateral membranes and T-tubules. 28 Intercalated discs (IDs) are highly coordinated structures located between the ends of myocytes along their 20 μm breadth, and comprising both communication (gap junction) and anchoring complexes (desmosomes and adherens junctions). 29 Mutations or acquired diseases that disrupt ID components are known to contribute in arrythmogenesis. The second and third populations of Na V 1.5 channels reside at lateral membranes which also comprise invaginations called T-tubules. This population of sodium channels ensures propagation of electrical impulse both in longitudinal and transverse directions ( Figure 2). 28 The Na V 1.5 channel contains several binding motifs for auxiliary proteins which interact to regulate its intracellular transport, cellular localization, gating and degradation ( Figure 1). These auxiliary proteins can be broadly categorized as adaptor proteins (anchor Na V 1.5 channel to cytoskeleton for trafficking and targeting to specific compartments of plasma membrane), enzymes (modify Na V 1.5 by post-translational modifications) and regulatory proteins (modulate gating). 9 Na V 1.5 ICL I-II contains several sites for phosphorylation and an interaction motif for a dimeric cytosolic adaptor protein 14-3-3η, a 30 kDa protein widely distributed in tissues of different organisms. Seven isoforms of the 14-3-3 protein are expressed in mammalian cells that interact with other protein partners by two consensus sequences known as "mode-1" (RXXpS/pTXP) and "mode-2" (RXXXpS/pTXP) binding motifs. 30 In cardiomyocytes 14-3-3η is co-localized with Na V 1.5 at the ID and its interaction with Na V 1.5 shifts steady-state inactivation towards hyperpolarization, with slowed recovery from inactivation. 31 Recently it has been shown that 14-3-3η facilitates Na V 1.5 channel α-α dimerization and mediates coupled gating. 32 These findings are quite intriguing given the widely held hypothesis that sodium channels exist as a single α-subunit in macromolecular complexes.
Ankyrin-G and a cofactor of Na V 1.5, multi-copy suppressor of GSP1 (MOG1), interact with ICL II-III . Both ankyrin-G and MOG1 are co-localized with Na V 1.5 at the ID. MOG1 is considered a cofactor for optimal expression of Na V 1.5, 33 while ankyrin-G organizes trafficking of Na V 1.5 at IDs and T-tubules. 34 Ankyrin-G is known to interact via the residue sequence 1047-VPIAVAESD-1055 in ICL II-III , 34 and coordinates a functional macromolecular complex at IDs with Na V 1.5, β IV -spectrin and CaMKII. 35 β IV -spectrin interacts with the C-terminal domain of

T-Tubule
Intercalated disc Lateral membrane

Cell 2
CaMKIIδ aMKIIδ Plakophilin2 Na V 1.5 αActinin2 CaMKIIδ and transgenic mice lacking this interaction show reduced levels of CaMKIIδ at intercalated discs, while levels in T-tubules remain unaffected, indicating distinct trafficking mechanisms at each subcellular compartment. Recruitment of CaMKII by β IV -spectrin at IDs in the vicinity of Na V 1.5 is followed by sodium channel phosphorylation, while disruption of this interaction, as observed in qv 3J transgenic mice, results in an increased I Na , rightward shift in fast inactivation and decreased late I Na . 36 Cardiospecific ankyrin-G knockout mice exhibit reduced I Na , decreased expression and localization of Na V 1.5 specifically at IDs of cardiomyocytes. At the same time, expression and localization of CaMKIIδ and β IV spectrin to IDs was decreased resulting in disruption of late I Na regulation by ankyrin-G, β IV spectrin, CaMKIIδ and the Na V 1.5 macromolecular complex. 28,35 Ankyrin-G also interacts with plakophilin-2 and connexin43 (desmosomal and gap junction proteins), 37 and is thus apparently an important player in coordinating mechanical and electrical signalling at IDs in cardiomyocytes ( Figure 2). 38 ICL III-IV is known to interact with α-actinin 2 which is an F-actin cross-linking protein. Both proteins co-localize at T-tubules and the interaction increases surface expression of Na V 1.5 without any changes in channel gating. 39,40 The last three amino acids (SIV) of the Na V 1.5 C-terminus constitute a PDZ domain binding site which interacts with syntrophins, synapse associated protein 97 (SAP 97 ) and protein tyrosine phosphatase 1 (PTPH 1 ). 41 Syntrophins form a macromolecular complex with dystrophins and coordinate localization of Na v 1.5 at lateral membranes ( Figure 2). 42 Dystrophin knockout mice exhibit conduction defects and reduced Na V 1.5 expression, 42 while SAP 97 together with ankyrin-G, ensures correct surface expression of Na V 1.5 at IDs. 28,39 Genetically modified Na V 1.5 (ΔSIV) mice, in which binding of syntrophin and SAP 97 has been disrupted, demonstrate downregulation of Na V 1.5 channels at lateral membranes, with slowed cardiac impulse conduction. 43 PTPH 1 interaction affects Na V 1.5 gating by shifting the availability curve towards hyperpolarized potentials. 44 A calmodulin (CaM) binding IQ-motif is also present at the C-terminus of the Na V 1.5 channel. 45 CaM is a small ubiquitous 17 kDa protein which binds/senses calcium ions. Its interaction with Na V 1.5 by binding through the IQ-motif results in enhancing the slow inactivation process and a hyperpolarizing shift in the I-V curve. 45,46 Recently, two additional interaction sites in ICL III-IV have been reported for CaM which modulates the Na V 1.5 channel by destabilizing the inactivated state and promoting faster recovery from inactivation. 47 The size of the Na V 1.5 channel population is determined by a balance between synthesis and degradation. An ubiquitin protein ligase (NEDD 4-2 ) binding pY-motif has been identified at the C-terminus of the Na V 1.5 channel. 48 E 3 ubiquitin-protein ligase NEDD 4-2 ubiquitinates the Na V 1.5 channel, thus giving the signal for internalization and finally degradation. 48 Co-expression of NEDD 4-2 with Na V 1.5 in Xenopus oocytes, decreased ionic currents by up to 40-65% with significant reduction in membrane expression. Conversely, an inactive NEDD 4-2 analogue increased both I Na and Na V 1.5 membrane expression. 48,49 Several modulators such as 14-3-3 protein, MAPKs, PKA and serine/threonine kinase SGK regulate NEDD 4 . 27,50 Recently, αB-crystallin was reported to interact with Na V 1.5 via ICL II-III and C-terminus. αB-crystallin co-localizes with Na V 1.5 and increases I Na by decreasing the ubiquitination. 51 Additionally, fibroblast growth factor homologous factors like FGF 12 , FGF 13 and FGF 14 also interact with the C-terminus of Na V 1.5 affecting the expression, trafficking and gating of the channel. 52,53 Lastly, the role of the N-terminus of Na V 1.5 is not fully understood yet; however, certain missense mutations in the N-terminus lead to degradation of the channel and exert a negative effect on wild-type (WT) channels. Also co-expression of the N-terminus peptide fragment with WT channels resulted in a twofold increase in surface expression and I Na compared to WT channel alone. This indicates that α-subunits of Na v 1.5 channel interact via the N-terminus by unknown mechanisms. 54 Recently, it has been shown that an internal PDZ-like domain, present in the N-terminus of Na V 1.5 interacts with α 1 -syntrophin, exerting a chaperonelike effect to positively modulate Na V 1.5, Kir2.1 and Kir2.2 channels in cardiomyocytes. 55 Moreover, several phosphorylation sites for Fyn kinase have also been identified in the N-terminus which may contribute to a depolarizing shift of fast inactivation, produced by Fyn kinase interaction with Na V 1.5 channel. 56 Caveolin-3, desmoglein-2, glycerophosphoryl diester phosphodiesterase-like protein 1 (GPDL 1 ), plakophilin-2, telethonin and Z-band alternatively spliced PDZ-motif protein (ZASP), are also reported to interact with Na V 1.5 channel through unidentified sites ( Figure 1). 39 The specific purpose of these distinct pools is not well established, but based on recent studies the proportions of current generated by these pools can be speculated. Sodium channels present on T-tubules generate around 20%, while Na V 1.5 channels residing on lateral membranes about 30% of I Na . Thus, the remaining 50% of I Na may be attributed to Na V 1.5 channels localized on IDs. 28

| MODULATION OF KINETICS
AND TRAFFICKING OF NA V 1.5 CHANNEL BY PHOSPHORYLATION 4.1 | PKA PKA, first described in 1968, 57 is a well-studied protein kinase. It is a holoenzyme existing as a heterotetramer with two catalytic and two regulatory subunits. When a second IQBAL AND LEMMENS-GRUBER messenger cAMP binds to these regulatory subunits, a conformational change takes place which releases and activates the catalytic subunits. 58 PKA is targeted to different sub-cellular locations by a scaffolding protein called A-kinase anchoring protein (AKAP), where upon activation, it phosphorylates the target proteins by transferring γ-phosphate of ATP. 59 PKA phosphorylates myriad of proteins including the Na V 1.5 channel. 60 The generalized consensus motifs in substrate proteins for phosphorylation by PKA include R/KXS/T, RRXS/T and R/KXXS/T. 61 Initial evidence for PKA mediated modulation of the Na V 1.5 channel was observed by stimulation of the β-adrenergic (β-AR) system with isoproterenol which decreased I Na and produced a hyperpolarizing shift in steady state inactivation by increasing levels of cAMP. 62,63 Both decreased upstroke velocity of action potential and decreased I Na has been reported in neonatal rat and adult guinea pig ventricular myocytes through PKA and Gprotein-regulated pathways. [62][63][64][65] Contrarily, increased I Na has also been reported in guinea pig myocytes, 66 rabbit myocytes 67 and Na V 1.5 expressed in Xenopus oocytes. 68 This increase in I Na was attributed either to phosphorylation of Na V 1.5 by PKA or activation of G-protein (Gsα), which was observed at hyperpolarized potentials (negative to -75 mV) without any effect on Na V 1.5 channel gating kinetics. 67 Some possible explanations for these initial conflicting reports include different voltage protocols used (depolarized vs hyperpolarized holding potentials), 62 or different concentrations of isoproterenol used in different experimental settings. 69,70 This discrepancy in I Na by cAMP or β-AR stimulation was further elaborated in experiments showing a test pulse of -50 mV at a holding potential of -150 mV increased I Na , while a test pulse of +30 mV at a holding potential of -90 mV decreased I Na . However, no effect was observed when sodium currents were elicited by a test pulse of +30 mV at a holding potential of -150 mV. 71 Later, with the help of biochemical studies, it was demonstrated that PKA phosphorylates serine residues at positions 526 and 529 in ICL I-II of the rat Na V 1.5 channel. However, no functional data for the biophysical characteristics of Na V 1.5 channel for these phosphorylated sites were provided by the authors. 72 The involvement of ICL I-II in modulation by PKA, was also confirmed in human Na V 1.5 channel (hH1 variant), where injection of cAMP in Xenopus oocytes expressing Na V 1.5 channel increased the conductance of channel without affecting half maximal activation or inactivation. 73 The increase in I Na by PKA stimulation develops slowly without reaching saturation over a time period of one hour, suggesting the involvement of an additional mechanism other than direct modulation of the channel by phosphorylation. 74 Whole cell patch clamp experiments in rat myocytes demonstrated that the increased I Na observed upon β-AR stimulation was due to the increased number of functional sodium channels, rather than activation by PKA and Gsα, which does not affect gating or open probability of the channel. 75 Further evidence for the involvement of PKA in Na V 1.5 channel trafficking was obtained by incubating Na V 1.5 channel expressing cells with chloroquine and monensin. Both the drugs interfere in recycling of membrane proteins and pre-incubation with these two drugs does not increase I Na on PKA stimulation. 74 These reports clearly indicate that PKA mediated phosphorylation increases I Na by promoting forward trafficking and increasing total number of functional Na V 1.5 channels. 76,77 In the rat Na V 1.5 channel, the involvement of two serine residues at positions 526 and 529 was reported earlier to increase I Na upon PKA activation. 72 Mutation of these conserved serine residues at positions 525 and 528 to alanine ( Figure 3; Table 1), in human Na V 1.5 channel (hH1 variant), abolished the PKA-mediated increase in I Na suggesting the same mechanism may also play a role in modulation of other Na V 1.5 channel isoforms. 78 In ICL I-II there are three endoplasmic reticulum (ER) retention signals (RXR), R 479 KR 481 , R 533 RR 535 and R 659 QR 661 which are either up-or downstream of the PKAphosphorylated serine residues. Addition of a phosphate group on serine 525 or 528 imparts large negative charge on these residues which may mask the ER retention signals, thus promoting forward trafficking of the Na V 1.5 channel. ER retention signals play an important role in trafficking of membrane proteins, since newly formed proteins are retained in the ER and released when these ER signals are masked by binding of another protein. 78 Among the three ER-retention sites, the R 533 RR 535 site plays a major role in PKA-mediated increased I Na ; it could be argued, that phosphorylation of serine 525 and 528 could mask this site and promote forward trafficking of the Na V 1.5 channel. 78 This forward trafficking redistributes Na V 1.5 channels from intracellular reservoirs like the ER and caveolae in the plasma membrane, thus increasing Na V 1.5 channel recruitment up to 45%. 77 PKA is a downstream effector of the β-AR signalling pathway and plays an important role in cardiac excitationcontraction coupling. 79 In cardiomyocytes, β-AR couples with stimulatory Gs protein and stimulation of the adrenergic system produces cAMP which activates PKA. β-AR stimulation modulates Na V 1.5 channel directly by PKAmediated phosphorylation or indirectly by Gs α signalling pathway. 75 PKA activation can fractionally increase I Na in epicardial border zone cardiomyocytes of infarcted canine heart. 80 Moreover, a missense mutation (R 526 H) in ICL I-II of Na V 1.5 channel resulted in a BrS phenotype with markedly reduced I Na . This mutation resides in the PKA recognition site which inhibits PKA-mediated phosphorylation of the Na V 1.5 channel, resulting in reduced incorporation of functional channel into the plasma membrane. Activation of PKA does not increase the I Na by rescuing R 526 H mutant channels, which also underpins the importance of PKA-mediated phosphorylation of the channel. 81 Apart from the Na V 1.5 channel, PKA also phosphorylates Ca V 1.2, phospholamban, troponin I and C, ryanodine receptors and several other proteins, and so plays important role in cardiac physiology. 82 However the direct involvement of PKA in cardiac pathophysiology or the use of PKA to improve cardiac pathology is not well described; further studies are required to characterize the PKA-Na V 1.5 interaction as a potential target for drug discovery and to reveal its potential role in cardiac pathophysiology.

| PKC
PKC consists of a single polypeptide with a regulatory Nterminus and catalytic C-terminus. This family of kinases transduces a diverse range of signals and since the identification of the first member in the 1980s, 11 different isozymes have been identified and categorized into three classes: calcium-dependent conventional PKCs (α, β I , β II and γ), calcium-independent novel PKCs (δ, ε, η, θ and μ) and atypical PKCs (ζ and λ). 83 PKC is a downstream effector of several circulating hormones such as angiotensin II (Ang-II), endothelin and norepinephrine, which upon stimulation cause phosphorylation of several cardiac proteins, activate other kinases and alter gene expression. These processes influence impulse conduction and EC-coupling implicated both in normal physiology and pathological conditions. 84 Like PKA, PKC phosphorylates serine and threonine residues in substrate proteins, but compared to PKA it displays less specificity. 85 Initially it was reported that PKC activators like TPA (phorbol ester) and 1,2-dioctanoglycerol (diacylglycerol analogue) increased single channel sodium currents and rate of current decay in neonatal rat F I G U R E 3 Phosphorylation of Na V 1.5 channel. Diagrammatic presentation of phosphorylation of the Na V 1.5 channel α-subunit at serine, threonine and tyrosine residues by PKA, PKC, CaMKII and Fyn kinase with respective colours. The amino acids numbering is according to Na V 1.5c isoform (Uniprot identifier # Q14524-1), while the amino acids with asterisk signs 113 correspond to Na V 1.5 hH1c isoform numbering T A B L E 1 Modulation and phosphorylation of Na V 1.5 channel. Phosphorylation of Na V 1.5 channel serine, threonine and tyrosine residues by PKA, PKC, CaMKII, Fyn and SGK with the method of identification and the most commonly reported effects on Na V 1. ventricular myocytes. 86 Later it was shown that activation of PKC by OAG (1-pleoyl-2-acetyl-sn-glycerol) decreases I Na and creates a hyperpolarizing shift in steady-state inactivation both in neonatal rat ventricular myocytes and rat cardiac sodium channel (rNa V 1.5), stably expressed in the Chinese hamster lung 1610 cell line. This decrease in I Na was attributed to a decreased open probability in singlechannel studies. 87 Subsequently, the same authors reported that serine 1505 in ICL III-IV was involved in modulation of rNa V 1.5 by PKC; upon replacement of this serine by alanine, PKC activation did not decrease I Na nor did it mediate a hyperpolarizing shift in fast inactivation. 88 This serine residue is conserved in several sodium channel isoforms and plays a role in modulation by PKC; however, the authors did not provide any direct evidence for phosphorylation of serine 1505. Similarly, in Xenopus oocytes transiently expressing human Na V 1.5 (hH1 variant), activation of PKC by PMA (phorbol 12-myristate 13-actate) or OAG decreased I Na without any hyperpolarizing shift in steadystate inactivation. The decrease in I Na was in part attributed to phosphorylation of serine 1503 (homologous to serine 1505 in rNa V 1.5) in ICL III-IV because mutation of this serine did not completely abolish the effect of PKC activation ( Figure 3; Table 1). 89 PKC activation by phorbol esters or diacylglycerol derivatives exhibit diverse effects on cardiomyocytes. These agonists are nonspecific and have multiple targets besides PKC which may also exert nonspecific effects on Na V 1.5 channel. To investigate these differential effects a peptide PKCP was used to specifically activate endogenous PKC and observe effects on the Na V 1.5 channel in rat ventricular myocytes. This peptide blocks the auto-regulatory region of native PKC and exposes the catalytic site to bring about phosphorylation of substrate proteins. Activation of PKC by PKCP peptide caused a dose dependent depolarizing shift in half maximal inactivation of the Na V 1.5 channel, while no effect was observed on half maximal activation or peak I Na . This depolarizing shift in inactivation was reversed by PKC inhibitors such as chelerythrine chloride or saturosporine. Besides the depolarizing shift in the inactivation curve, PKC activation also slows I Na decay and channel inactivation, while recovery from inactivation is enhanced. 90 Human cardiomyocytes express nine different isoforms of PKCs irrespective of normal or diseased states; however, levels of these isoforms may vary in different conditions. 84 PKC activators such as phorbol ester or diacylglycerol analogues are nonspecific in nature and it is highly likely that different PKC isoforms act differently providing a reason for differential effects reported in various studies of activation of PKC. 86,87,90 By using peptide activators or inhibitors of PKC isoforms it was described that specific activation of εPKC decreases I Na in rat ventricular myocytes and the human Na V 1.5 channel (hH 1 isoform) heterologously expressed in Xenopus oocytes, without affecting voltage dependence of activation or inactivation. However the blockade of εPKC did not completely abolish the effect of PMA which suggests the involvement of other PKC isoforms as evidenced in another study where activation of PKCα decreased the I Na . 91,92 With the development of specific PKC activators it was described that activation of PKCα decreased I Na without any effect on gating. This decrease in I Na was slow in development and non-saturable which was supposedly because of internalization or re-distribution of functional hNa V 1.5 channels away from the plasma membrane. 92,93 This modified intracellular trafficking of hNa V 1.5 channels was attributed to PKC-mediated phosphorylation of the channel and ROS. 93 Moreover, the authors also described that in pre-blocked cPKCs, low concentrations of PKC activators such as PMA (1 nM) and thymeleatoxin (50 nM) cross activate PKA which slightly increases I Na and explains initial conflicting results on PKC activation. 92 Renin-angiotensin signalling is known to activate several PKC isozymes and in several cardiovascular disorders this pathway is chronically activated. In transgenic mice overexpressing cardio-specific angiotensin-II type 1 receptors (AT1R), QRS complex widening and slower action potential was observed because of decreased I Na . This AngII-AT1R pathway stimulation activates PKCα and co-localizes it with Na V 1.5 at the plasma membrane where their interaction results in decreased I Na . 94 Recently a metabolic pathway for PKC activation has been described where the elevated levels of NADH activate PKCδ, resulting in a decrease in I Na . 95 Interestingly, this decrease in I Na was not because of any change in surface expression but rather channel conductance was decreased directly by phosphorylation of serine 1503 in Na V 1.5 channels and indirectly by increasing ROS production in mitochondria. 95,96 The PKCδ antagonism completely reversed both decrease in I Na and ROS production from the mitochondria while specific inhibition of PKCα could partially recover I Na without any effect on ROS production, thus indicating the involvement of more than one PKC isozyme. By mutational analysis it was described that both phosphorylation of serine 1503 in Na V 1.5 channels and ROS production in mitochondria are required for PKCδ-mediated modulation of the Na V 1.5 channel. 95 Alteration in sodium current is detrimental in underlying disease conditions and may trigger arrhythmias leading to sudden cardiac death. 94 As discussed above, activation of several PKC isozymes are reported to influence the I Na and the reported effects on Na V 1.5 are contradictory, which might be because of involvement of different PKC isozymes. Activation of PKC results in phosphorylation of a conserved serine residue in ICL III-IV which alters Na V 1.5 channel trafficking, but besides this serine residue phosphorylation of other serine residues is also likely. Additional detailed studies are warranted which might unravel further aspects of PKC signalling and may explain the differential effects reported on Na V 1.5 channel by PKC activation.

| CaMKII
CaMKII is a multifunctional enzyme class consisting of four isoforms. The αand βisoforms are mainly expressed in neuronal tissues while γand δare expressed ubiquitously. 97 Alternative splicing of primary transcripts results in generation of multiple variants. Six splice variants of the CaMKIIδ isoform have been identified in the heart. 98 As the name indicates, CaMKII activity is dependent on Ca 2+ and/or CaM, which after binding induces conformational changes in CaMKII to autophosphorylate at threonine 287. Autophosphorylation keeps CaMKII active after dissociation of Ca 2+ /CaM. 99 CaMKII is also activated in a Ca 2+ independent manner by ROS-mediated oxidation of methionine residues at positions 281/282. This mode of activation requires initial binding of Ca 2+ /CaM to expose the potential methionine residues followed by oxidation. Oxidative stress is observed in HF, after myocardial infarction (MI) and with increased levels of Ang-II. 100 Moreover levels of CaMKIIδ C are also increased during pressure overload, HF and sustained β 1 -AR stimulation thus both factors contributing in outcomes of adverse cardiac events. [101][102][103] Hence, CaMKII Inhibition appears to be cardio-protective after MI by reducing apoptosis and remodelling associated with excessive stimulation of β-AR signalling and Ang-II. 100,104 Initial evidence for CaMK-mediated modulation of Na V 1.5 channel was obtained indirectly using CaMK inhibitors such as KN-93 and autocamtide-2 related inhibitory peptide (AIP). KN-93 slowed Na V 1.5 channel current decay, produced a depolarizing shift in fast inactivation and slowed entry into inactivated states. Interestingly, AIP the specific inhibitor of CaMKII did not exhibit any effects on Na V 1.5 channels and the authors suggested the involvement of CaMKIV in modulation of Na V 1.5 channels. 105 Direct evidence for CAMKII interaction with Na V 1.5 channel was obtained from transgenic mice expressing CaM-KIIδ C and in rabbit ventricular myocytes with acute overexpression of CaMKIIδ C . Overexpression of CaM-KIIδ C increased the phosphorylation of Na V 1.5 channels and in both experimental models CaMKIIδ C interaction slowed Na V 1.5 channel fast inactivation, created a hyperpolarizing shift in steady-state inactivation, increased late I Na and number of Na V 1.5 channels undergoing intermediate inactivation along with slowed recovery from inactivation. Together these effects could be arrhythmogenic (prolonged QT and QRS intervals) as has been observed in transgenic mice overexpressing CaMKIIδ C . 106 CaMKII inhibition by KN-93 reduced late I Na in ventricular myocytes from normal and experimental canine model of chronic HF. 107 CaMKII inhibition by KN-93 under basal physiological conditions in rat ventricular myocytes reduced peak I Na and late I Na , produced a hyperpolarizing shift in fast inactivation, and augmented intermediate inactivation with slowed recovery from both fast and slow inactivation. It also reduced membrane excitability by decreasing upstroke velocity during action potential. 108 These results are not consistent with overexpression of CaMKII, 106 which caused a hyperpolarizing shift in inactivation and slowed recovery from inactivation. In guinea pig ventricular myocytes CaMKIIα increased peak I Na , produced a depolarizing shift in fast inactivation, accelerated recovery from inactivation, increased late I Na and decreased the fraction of channels undergoing intermediate inactivation. CaMKIIα also increased action potential duration while blockade by KN-93 shortened it. Moreover, CaMKIIα-mediated phosphorylation was observed in the ICL I-II and C-terminal domains of the Na V 1.5 channel. 46 These results differ in certain aspects from rabbit ventricular myocytes overexpressing CaMKIIδ C , which demonstrated hyperpolarizing shift in fast inactivation, slowed recovery from inactivation and increased intermediate inactivation. 106 These differences can be accounted for by different methodological approaches, different CaMKII (α vs δ) isoforms, altered phosphorylation of Na V 1.5 channel due to overexpression of CaMKIIδ C and association of the chronic overexpression of CaMKIIδ C with cellular and structural remodelling. The effects of CaMKII on Na V 1.5 remained disputed; however, there is a general consensus on increased late I Na , but sodium channel gating and kinetics are debatable. Late I Na is increased in certain pathological conditions like LQTS-3, cardiac ischemia or HF. Increased late I Na overloads cellular calcium and sodium levels, which may play a key role in diastolic dysfunction and arrythmogenesis. Late I Na prolongs action potential duration by reducing repolarization reserves and may trigger early after depolarizations, while sodium and calcium overload may lead to delayed after depolarizations both in atrial and ventricular myocytes. 109 Pathological or drug-induced, increased late I Na elevates Na + levels in cardiomyocytes, which in turn elevates Ca +2 levels by sodium calcium exchanger thus paradoxically activating CaMKII. 21,23,24,110 Inhibition of late I Na by ranolazine prevents CaMKII activation and acts as a cardio-protective agent. 111 CaMKII interaction with Na V 1.5 in myocytes has been established by co-immunoprecipitation experiments and immunostaining which indicates co-localization of CaMKII and Na V 1.5 channels at IDs and T-tubules. 36,106,108 These co-immunoprecipitation experiments and co-localization of CaMKII with Na V 1.5 channels strongly suggest direct interaction of CaMKII with the Na V 1.5 channel. There are several CaMKII-mediated phosphorylation motifs (RXXS/ T) in Na V 1.5 channel and alanine-scanning suggested phosphorylation of serine residue at position 571 which was also validated by site-specific antibodies. In transgenic qv 3J mice, decreased phosphorylation of serine 571 was observed following disruption of interaction of CaMKII with the Na V 1.5 channel. 36 Activated CaMKIIδ C interacted stably with ICL I-II of Na V 1.5, the loop which has been shown to contain more than one phospho-acceptor sites for CaMKII. In vitro phosphorylation of Na V 1.5 by CaMKIIδ C showed phosphorylation of serine 483/484, 516 and serine 593/threonine 594, but the authors 112 detected no phosphorylation on serine 571 35,36 as previously reported. Further biochemical tests established that only phosphorylation on serine 516, 593 and threonine 594 are involved in CaM-KIIδ C -mediated modulation of Na V 1.5 with preference order of 516 > 594 > 593. Alanine mutagenesis of serine 516, 571 and threonine 594, but not serine 593, prevented a CaMKIIδ C mediated hyperpolarizing shift in sodium channel availability and intermediate inactivation, indicating their phosphorylation-dependent role in regulation of Na V 1.5 by CaMKIIδ C . 112 Mass-spectrometry based analysis of in situ immunopurified Na V 1.5 channel revealed eleven phosphorylated residues, of which 10 reside in ICL I-II (serine 457, 460, 483, 484, 497, 510, 524/525, 571, 664, 667), and one resides in the N-terminus (serine 36/39/42/thre-onine38). Several interacting protein partners, including CaMKII -β, -δ, -γ subunits, CAM, and FGF-13 were also identified by mass-spectrometry based analysis from immunoprecipitated Na V 1.5 of mouse ventricular myocytes by the same authors. 113 Recently, nine more Na V 1.5 phosphorylated serine and threonine residues (486, 499, 516, 539, 1012, 1888, 1937, 1938 and 1989) have been reported by the same research group investigating WT and transgenic mice overexpressing CaMKIIδ C . Among these, phosphorylation of serine residues at position 1938 and 1989 was increased by CaMKIIδ C overexpression. Both serine residues (1938 and 1989) are conserved in the human Na V 1.5 channel and when the orthologous serine residues at position 1933 and 1984 in human Na V 1.5 (hH1c) are mutated, interaction of FGF-13 with Na V 1.5 channels is disrupted; this alters fast inactivation, increases late I Na and decreases channel availability. 114 In another recent study, mass-spectrometry based analysis of immunopurified and CaMKIIδ C -mediated in vitro phosphorylation of human Na V 1.5 expressed in HEK-293 cells revealed 31 serine and three threonine phosphorylated residues. 115 Fifty percent of these phosphorylation sites were located in ICL I-II , underpinning the importance of the first IC-loop in modulation of Na V 1.5 by post-translational modifications. Of these phosphorylated residues 17 were present at baseline while 23 residues were phosphorylated by CaMKIIδ C . Phosphorylated amino acids were more scattered in the ICL I-II , while they were clustered together in the N-and C-termini. 115 At the N-terminus, serine 11, 12, 20, 42 and 61 were phosphorylated by CaMKIIδ C , and of these, serine 42 was also phosphorylated at baseline while threonine 17 was phosphorylated by an unknown kinase. Eighteen phosphorylated residues were present in ICL I-II and seven of them (serine 460, 471, 484, 516, 528, 539, 571, 667 and threonine 455) were phosphorylated by CaMKIIδ C . Serine 460, 483, 484, 497, 510, 516, 577 and threonine 570 were also phosphorylated at baseline, while serine 457, 464, 499 and 664 were phosphorylated by an unknown kinase. ICL II-III and ICL III-IV contained one phosphorylated residue each, with serine 1003 and 1503, respectively. Eight phosphorylated residues were identified in the C-terminus and seven of them (serine 1865, 1885, 1920, 1925, 1934, 1937 and 1998) were phosphorylated by CaMKIIδ C . Two serine residues 1937 and 2007 were also phosphorylated at baseline ( Figure 3; Table 1). 115 The majority of these identified phosphorylation sites are not characterized functionally; however, several are well described in pathological processes. Secondly, it may be emphasized that the phosphorylation status can differ on the same residue under normal physiological or pathological conditions and the same residue can be phosphorylated by multiple kinases. 113 Phosphorylation at S516 is decreased in HF patients and it is also known that methylation at arginine 513 decreases phosphorylation of serine at position 516 indicating intriguing crosstalk between methylation and phosphorylation. [115][116][117] CaMKII-mediated phosphorylation of serine 571 specifically regulates the late I Na component of Na V 1.5 without affecting channel availability and recovery from inactivation. This serine 571 mediated increased late I Na prolongs APD and QT interval with increased susceptibility to arrhythmias. Ventricular myocytes from transgenic mice with hyperphosphorylation at position 571 show increased late I Na and β-AR stress induced afterdepolarizations with repolarization abnormalities. Moreover, phosphorylation at S571 contributes in HF, but not in pressure overload hypertrophy. 118 Two proarrhythmic Na V 1.5 variants (A 572 D and Q 573 E) were initially identified in extensive genetic analysis of LQTS probands. [119][120][121] Both of these variants reside near the CaMKII phosphorylation site serine 571. Although these variants do not have any effect on serine 571, this substitution introduces negative charge which simulates a phosphorylated residue. Both these variants exhibited increased late I Na which was reversed by ranolazine and produced hyperpolarizing shift in fast inactivation with slowed recovery from inactivation. Lengthening of action potential duration was also observed with afterdepolarizations, which were eliminated by ranolazine. 122 Effects produced by these variants recapitulate CaMKII mediated effects, and computational models indicate that both variants show structural and electrostatic features similar to the serine 571 phosphorylated channel. Phosphorylated CaMKII and CaMKII mediated increased phosphorylation of serine 571 was observed in ventricular myocytes of non-ischaemic HF patients and in canine post infarct border zone, but not in transgenic mice AC3-I expressing CaMKII inhibitor. 122 CaMKII-mediated Na V 1.5 channel phosphorylation and regulation exhibits both gain-of-function (increased late I Na ) and lossof-function (decreased availability) effects, indicating complex regulation by phosphorylation. Moreover, further detailed studies are required to link CaMKII-mediated phosphorylated residues with pathological process of cardiac disorders and their role in disease progression.

| Fyn kinase
Besides phosphorylation of serine and threonine, the Na V 1.5 channel is also modulated by phosphorylation of tyrosine residues. Initial evidence for tyrosine phosphorylation of the Na V 1.5 channel was reported indirectly by the use of protein tyrosine kinase inhibitors (genistein, AG957, PP2 and ST638), which all decreased I Na while genistein and AG657 also produced a hyperpolarizing shift in steady-state fast inactivation and prolonged recovery from inactivation. 123 Similarly, co-expression of PTPH1 with Na V 1.5 also shifts steady-state inactivation towards hyperpolarized potentials. 44 Fyn tyrosine kinase is a member of the non-receptor Src family of tyrosine kinases which is expressed ubiquitously. 124 Fyn kinase consists of four domains (SH 1 -SH 4 ). The SH 1 domain possesses tyrosine kinase activity and is located in the C-terminal domain. The SH 2 domain recognizes phosphorylated tyrosine while the SH 3 domain is non-catalytic and binds target proteins through prolinerich regions. 124 The generalized proline-rich recognition motifs of the SH 3 domain are categorized as class I or class II and have the sequence R/KXXPXXPX and PXXPXR/K, respectively. 125,126 The SH 4 domain constitutes a palmitoylation and myristoylation sequence for membrane anchoring and is located at the N-terminus. 124 In cardiomyocytes Fyn kinase regulates stability of adherens junctions and is also localized at caveolae along with Na V 1.5 channels. [127][128][129] Interaction of Fyn kinase with Na V 1.5 has been established where it co-immunoprecipitates and phosphorylates the Na V 1.5 channel. 130 This phosphorylation creates a depolarizing shift in steady-state fast inactivation, increases recovery from inactivation and decreases rate of entry into slow inactivation. 131 Interaction of Fyn kinase with Na V 1.5 is complex and involves multiple steps. Binding of Fyn kinase to proline-rich regions in the ICL I-II domain and C-terminus is followed by phosphorylation of nearby tyrosine residues in the N-terminus (Y 68 , Y 87 , and Y 112 ), ICL III-IV (Y 1494 , Y 1495 ) 130,131 domain and C-terminus (Y 1811 , Y 1889 ), indicating a complex and multistep modulation. 56

| Phosphoinositide 3-kinase signalling
Phosphoinositide 3-kinases (PI3K) are a group of kinases that phosphorylate the 3-hydroxyl group of inositol in phosphoinositides. They are categorized into three classes (class-1, class-2 and class-3) depending upon structure, subunits and substrate specificity. The mammalian heart expresses different isoforms from the three classes of PI3Ks but most of the studies have focused on describing the role of class-1 PI3Ks in cardiac electrophysiology. Class-1 PI3Ks are heterodimers consisting of a catalytic subunit (PI3Kα, PI3Kβ, PI3Kγ or PI3Kδ) bound to a regulatory subunit. 132 Specific inhibition of PI3Kα in mouse ventricular myocytes increased action potential duration by prolonging the QT interval, decreasing peak I Na and increasing the late I Na , while no effect was observed on the blockade of other three catalytic subunits. 133,134 The downstream effectors of PI3K pathway are protein kinase Akt/ PKB and 3-phosphoinositide dependent protein kinase 1 (PDK1). Downregulation of Akt is observed in diabetes as PI3K signalling is reduced due to decreased insulin levels which increase QT interval and late I Na . 135 Activation of PI3K translocates PDK1 to the plasma membrane which then activates atypical PKC isoforms and serum and glucocorticoid inducible kinase (SGK). 132 PDK1 is an important member of the AGC family of protein kinases and it acts an upstream protein kinase to several members of AGC family, including SGK. Mice with conditional knockout of PDK1 exhibit lower heart rate with QRS and QTc interval prolongation, due to reduction of peak I Na and reduced surface expression of sodium channels. Na V 1.5 channel gating was also changed with a moderate hyperpolarizing shift both in activation and inactivation curves. This decreased surface expression of Na V 1.5 channel was attributed to PDK1 mediated activation of Foxo1 pathway. 136 SGK is a serine and threonine kinase, which is transcriptionally regulated by gluco-and mineralocorticoids. It is a downstream target of PI3K pathway and phosphorylated by PDK1 which activates SGK. 137 Three isoforms of this kinase have been identified and named as SGK1, SGK2 and SGK3. Among these, SGK1 and SGK3 are expressed in cardiac tissues and interact with the Na V 1.5 channel. The co-expression of SGK1 or SGK3 with Na V 1.5 channel (hH1 variant) in Xenopus oocytes increases I Na and additionally SGK3 also creates a depolarizing shift in the inactivation curve and a hyperpolarizing shift in the activation curve. These effects have been attributed to the phosphorylation of two serine residues at positions 483 and 664 in the Na V 1.5 channel, identified by mutational analysis (Table 1). 138,139 SGK1 also controls sodium transport in the kidneys, thus acting as an important contributor in HF and arrhythmia. In pressure overload after transverse aortic constriction (TAC), its acute activation proves to be cardio-protective however its chronic activation in TAC induced HF acts conversely. Both systolic and diastolic dysfunction has been observed in transgenic mice expressing cardio-specific, catalytically active SGK1, while transgenic mice expressing the catalytically inactive variant of SGK1 showed normal cardiac structure and function. When additional stress was introduced by TAC, the baseline cardiac dysfunction in catalytically active SGK1 mice was markedly exacerbated, while WT and transgenic mice expressing catalytically inactive SGK1 better tolerated TAC, suggesting that SGK1 inhibition prevents fibrosis, cardiac hypertrophy and development of HF after pressure overload. Moreover, transgenic mice expressing the catalytically active variant of SGK1 exhibit ECG abnormalities, action potential prolongation along with spontaneous ventricular tachycardia. These effects were attributed to increased peak I Na , late I Na and changes in Na V 1.5 channel gating including a hyperpolarizing shift both in the activation and inactivation curves, thus increasing the window current. Surface expression of Na V 1.5 channels was also increased by SGK1 activation since SGK1 inhibits Nedd 4-2 , thus preventing Na V 1.5 channel internalization. 139 Recently, a threonine residue at position 1590 in the C-terminal domain of the Na V 1.5 channel has been described as a candidate site for phosphorylation by SGK1; replacing this threonine with alanine almost completely abolished SGK1-mediated increase in I Na . 140 Interestingly, PI3K-mediated effects on I Na appear to be opposite to that of its downstream effector SGK. Moreover Akt and SGK also behave oppositely, suggesting that phosphorylation of the Na V 1.5 channel is the underlying cause of this differential modulation. PI3K pathway inhibition prolongs QT interval as observed with nilotinib which increases QT interval through inhibition of PI3K pathway. 133 Besides the Na V 1.5 channel, PI3K signalling affects several cardiac ion channels, 132 rendering description of its effect on cardiac electrophysiology complicated and warranting further studies to describe its specific role.

| CONCLUDING REMARKS
Cardiac disorders such as structural heart diseases and arrhythmogenic conduction defects are major public health problems both in developed and developing countries. Use of ion channel blockers can treat arrhythmias but still cannot reduce mortality rates, thus stressing the need for new suitable treatment options. An appealing approach which has emerged recently is antagonizing the protein kinases implicated in pathology of the cardiac disease. One such example is CaMKII which is upregulated in HF and hypertrophy and pharmacological inhibition of which in experimental animal models has proven to be cardio-protective in a setting of arrhythmias and HF. Kinases and their mediated pathways are also gaining attention as biomarkers for early detection of cardiac disorders. Phosphorylation and IQBAL AND LEMMENS-GRUBER | 13 of 18 dephosphorylation reactions are carried out by more than 500 kinases and 100 phosphatases. Mass-spectrometry based proteomic studies are unveiling information regarding phosphorylated residues but still a lot of information is missing regarding the sites, sequences, order of phosphorylation, cross talk between the modified residues and how these complicated events unfold to modulate physiological or pathological processes. This missing information strongly requires a concerted effort in the form of future studies to address these questions in order to understand these pathways for the discovery of new drug targets.