Ubiquitin-Protein Ligases of the Nedd4/Nedd4-like Family

Ubiquitylation of target proteins tags them for proteasome- or lysosome-dependent degradation²³ but also serves multiple nondegradative functions, in particular the trafficking of membrane proteins.²⁴ Ubiquitin is a small protein of 76 amino acids found in all animal cells.²⁵ It binds covalently to lysine residues of target proteins. This protein ubiquitylation is performed by E3 ubiquitin-protein ligases.²⁵ Ubiquitylated membrane proteins are subsequently internalized and can be targeted for lysosomal or proteasomal degradation. Alternatively, they can also be deubiquitylated by specific proteases and recycled back to the membrane.^26,27 Many different ion channels have been reported recently to be regulated by the Nedd4-like family of E3 ubiquitin-protein ligases. Nedd4-like enzymes bind specifically to target proteins that have consensus domains known as PY motifs with the sequence [L/P]PxY.²⁸ Such PY motifs are found in the C-termini of all voltage-gated Na⁺ channels (with the exception of Na_v1.4, Na_v1.9, and Na_x^29,30), as well as in other cardiac ion channels.^27,31,32 Nedd4/Nedd4-like enzymes harbor several WW domains³³ that can interact with these PY motifs. When expressed in Xenopus oocytes, Na_v1.5-mediated I_Na is decreased by Nedd4-2–mediated ubiquitylation.³⁴ Our group investigated the molecular determinants of this regulation³⁵ and found that the ubiquitin-protein ligase Nedd4-2 binds directly to the PY motif of Na_v1.5 and ubiquitylates the channel in mammalian cells. Because there was no reduction in the total level of Na_v1.5 protein upon coexpression with Nedd4-2, we concluded that this was most likely caused by an increased internalization rate rather than by Na_v1.5 degradation.²⁹ Inhibition of the proteasome has been linked recently to an increase in I_Na and Na_v1.5 expression in neonatal rat cardiomyocytes.³⁶ Ubiquitylated Na_v1.5 was found to be present in mouse cardiac tissue,³⁵ further suggesting that membrane turnover or the stability of Na_v channels can be regulated in vivo via ubiquitylation. Nine of such Nedd4-like E3 enzymes are present in the human genome,³⁷ at least eight of which are expressed at the RNA level in human cardiac tissue.³¹ It remains to be determined which of these Nedd4/Nedd4-like proteins regulates Na_v1.5 in a physiologic context and whether the PY motif plays a role in cardiac disease. Altogether these results suggest that the ubiquitin-proteasome system is involved in several aspects of Na_v1.5 regulation, the intricacies of which remain to be elucidated.

14-3-3η (eta) Protein

The 14-3-3 protein family is composed of dimeric cytosolic adaptor ubiquitous proteins.³⁸ The members of this family are involved in many cellular functions such as the binding and regulation of trafficking of various membrane proteins.³⁹ Allouis et al. performed yeast two-hybrid and coimmunoprecipitation experiments⁴⁰ showing that the isoform 14-3-3η interacts with the N-terminal part of the intracellular loop linking domains I to II (see Figure 18-1, A and Table 18-1). Furthermore, it was observed that 14-3-3 and Na_v1.5 are colocalized at the intercalated discs of myocytes. No influence on the Na_v1.5-mediated peak current was observed when Na_v1.5 and 14-3-3η were coexpressed in COS cells, suggesting that this protein does not influence Na_v1.5 trafficking. In this expression system, 14-3-3 shifted the inactivation curve toward negative potentials and delayed recovery from inactivation, illustrating that 14-3-3 proteins are able to modify the biophysical properties of ion channels. Because different isoforms of 14-3-3 proteins are expressed in cardiac cells,⁴⁰ their exact roles in normal cardiac function and their implications in disease states require further investigation.

Fibroblast Growth Factor Homologous Factors

Although fibroblast growth factor homologous factor (FHF) family members are highly homologous in sequence and structure to fibroblast growth factors (FGF), their functional roles are quite different. FHFs are found in the cytosol because their N-termini lack signal sequences that are required for secretion.⁴¹ The FGF homologous factors FHF3, FHF1, FHF2, and FHF4 are also called FGF11, FGF12, FGF13, and FGF14, respectively. One of the best characterized functions of the FHFs is their ability to bind to and modulate voltage-gated Na⁺ channels. Liu et al.⁴² were the first to demonstrate that FGF12 interacts with the proximal portion of the C-terminus of Na_v1.5 (see Figure 18-1, A). This finding was then confirmed by Goetz et al.,⁴³ who also showed that FGF11 and FGF14 interact with the same domain of Na_v1.5. In HEK293 cells, coexpression of FGF12 with Na_v1.5 shifted the steady-state inactivation relationship toward hyperpolarized values without affecting the other parameters studied.⁴² Interestingly, several LQTS type 3 and BrS mutations are located in the domain that interacts with FHF1B (see Table 18-1). The SCN5A mutation D1790G⁴⁴ disrupted the binding of this protein with Na_v1.5 and abolished the FHF 12-induced shift of steady-state inactivation. Another member of the FGF family, FGF14, has been shown to downregulate the activity of Na_v1.5.⁴⁵ Because this regulatory protein is expressed in the brain, and not in the heart,⁴⁵ the significance of this interaction is likely restricted to the central nervous system. A recent study⁴⁶ demonstrated that FGF13 is expressed in mouse cardiac myocytes. FGF13 was shown to directly bind to and colocalize with Na_v1.5 in mouse cardiac cells. Reducing the expression of FGF13 decreased the Na⁺ current, shifted the availability curve toward more negative potentials, and slowed recovery from inactivation. Furthermore, decreased membrane expression of Na_v1.5 was observed in cells with reduced expression of FGF13. These results underscore the specific role of FGF13 in modulating Na_v1.5 function and suggest that this gene may be a susceptibility gene for cardiac arrhythmias. A crystal structure of the Na_v1.5 C-terminal domain complexed with FGF13 and calmodulin was recently published.⁴⁷ The functional relationship between FGFs and calmodulin remains to be studied in more detail.

Calmodulin

Intracellular Ca²⁺ has been shown to modulate the function of many ion channels, including the voltage-gated Na⁺ channels.48,49 Many cardiac ion channels use calmodulin (CaM), a ubiquitous intracellular Ca²⁺-binding protein involved in many different cellular processes,⁵⁰ as a Ca²⁺-sensing partner. The Na_v1.5 C-terminal domain has an IQ motif with a consensus sequence of IQxxxRxxxxR (see Table 18-1), which is very similar to that found in voltage-gated calcium channels.⁵¹ The IQ motif is also found in all isoforms of the Na_v family.⁵² Several studies^47,53–55 have shown a direct interaction between CaM and the IQ motif of Na_v1.5. Recent work by Wang et al. described a crystal structure with an IQ motif ternary complex of Na_v1.5, FGF13, and CaM. The functional consequences of this CaM-Na_v1.5 interaction are controversial. Several studies^52,56,57 have shown inconsistent results that have been difficult to reconcile. A few groups^53,55–57 reported that the voltage dependence and stability of the inactivated state were dependent on CaM and the IQ motif. In addition, it has been proposed that CaM may not be the only sensor for the Ca²⁺-dependent regulation of Na_v1.5, because the proximal part of the Na_v1.5 C-terminal domain is similar to an EF-hand motif, which is known to bind Ca²⁺.^58,59 Whether this domain binds intracellular calcium is still under debate.^47,60–62 The role of intracellular Ca²⁺ in Na_v1.5 regulation was recently investigated by Casini et al.⁶³ They demonstrated that Na_v1.5 single-channel conductance is decreased (most likely directly) with an increase in the intracellular Ca²⁺ concentration. Intracellular Ca²⁺ appears to be an important regulator of Na_v1.5 function, but more studies are needed to clarify its role.

Ca²⁺/Calmodulin-dependent Protein Kinase II

Ca²⁺/calmodulin-dependent protein kinases II (CaMKII) are serine/threonine protein kinases expressed in many cell types, where they transduce intracellular Ca²⁺ increases into the phosphorylation of target proteins, including cardiac ion channels.⁶⁴ CaMKIIδc is the predominant cardiac isoform upregulated in human and animal heart failure models.^65,66 Na_v1.5 has been found to colocalize and coimmunoprecipitate with CaMKIIδc.^67–69 Ashpole et al.⁶⁹ have shown that CaMKIIδc interacts with the first intracellular loop of Na_v1.5 (see Figure 18-1) and that the residues Ser-516 and Thr-594 may be phosphorylated by this kinase. Hund et al.⁷⁰ found that Ser-571 was also a target of CaMKII within the same intracellular loop of Na_v1.5, and that this regulation was dependent on the correct expression of βIV-spectrin, which interacts with ankyrin-G at the intercalated discs. Overexpression of the CaMKIIδc enzyme in rabbit myocytes and transgenic mice induced a Ca²⁺-dependent hyperpolarizing shift of the steady-state inactivation curve, slowed the recovery from inactivation, and increased the persistent I_Na. These biophysical property alterations of I_Na are similar to some congenital LQTS type 3 mutation-dependent modifications.⁷¹ In mice, transgenic overexpression of CaMKIIδc leads to chronic heart failure and episodes of ventricular tachycardia. In dog ventricular myocytes, it has also been shown that CaMKII activity increases the persistent/late I_Na,⁷² but it is unclear whether these arrhythmias are the direct consequence of the Na_v1.5 biophysical alterations or are related to other heart failure mechanisms. Nevertheless, CaMKII is an important component in the pathogenesis of arrhythmias and potentially a new drug target.⁶⁴

Proteins Tyrosine Phosphatase PTPH1

Ion channels are also regulated by the phosphorylation of tyrosine residues, a process that depends on both the phosphorylation activity of tyrosine protein kinases and the dephosphorylation activity of phosphatases.⁷³ It was demonstrated that the overexpression of the protein tyrosine kinase Fyn in HEK293 cells altered several of the biophysical properties of Na_v1.5 via the phosphorylation of Tyr-1495.⁷⁴ This residue is close to the Ile-Phe-Met cluster (IFM) in the intracellular loop linking domains III-IV (see Figure 18-1, A), which is known to mediate the rapid inactivation process of voltage-gated Na⁺ channels.⁷⁵ Fyn coexpression in HEK293 cells shifted the steady-state inactivation curve toward depolarized potentials and accelerated the recovery from Na_v1.5 inactivation.⁷⁴ The site of interaction of Fyn with Na_v1.5 remains to be investigated. We reported⁷⁶ that the protein tyrosine phosphatase, PTPH1, interacts with the PDZ-domain binding motif of Na_v1.5, which is also known to interact with two other PDZ-bearing proteins, syntrophin⁹ and SAP97.⁴ PTPH1 coexpression in HEK293 cells shifted the availability curve of wild-type Na_v1.5 toward hyperpolarized potentials. This effect was abolished when the PDZ domain–binding motif of the Na_v1.5 C-terminus was removed by an early stop mutation. These results suggest that tyrosine phosphorylation of Na_v1.5 modulates the stability of the inactivated state. The fact that several proteins (e.g., PTPH1, syntrophin proteins and SAP97) interact with the same binding domain supports the coexistence of different multiprotein complexes with Na_v1.5 in cardiac cells.

Telethonin

Telethonin is a small 19-kDa protein expressed in striated muscle cells, including cardiac cells. Mutations in the gene coding for telethonin, TCAP, lead to hypertrophic and dilated cardiomyopathy and limb-girdle muscular dystrophy.77,78 This protein has been shown to interact with the sarcomeric protein titin,⁷⁹ as well as the β-subunit of the KCNQ1 channel (KCNE1), which mediates the cardiac IKs current.⁸⁰ Telethonin can be coprecipitated with Na_v1.5 from mouse cardiac tissue, and the two proteins colocalize in cardiomyocytes.⁸¹ The site of interaction on Na_v1.5 has not yet been mapped. When the endogenous expression of telethonin in HEK293 cells was reduced, the voltage-dependent activation of the Na_v1.5-mediated current shifted toward positive potential values. In addition, coexpression experiments of telethonin and Na_v1.5 in HEK293 cells altered several of the kinetic properties of the channel.⁸¹ Furthermore, a point mutation in TCAP was found in a patient with abnormal gut motility,⁸¹ consistent with the fact that Na_v1.5 and telethonin are expressed in the neurons of the intestinal mucosa.⁸² The precise role of telethonin in the regulation Na_v1.5 in the heart (and other tissues) remains to be clarified.

α–Actinin-2

α–Actinin-2 is a protein of the F actin–cross-linking protein family (similar to spectrin and dystrophin) that interacts with and regulates the trafficking of several potassium channels,⁸³ in particular Kv1.5. A recent study used⁸⁴ pull-down experiments to show that α–actinin-2 interacts with the intracellular III-IV loop of Na_v1.5. Coexpression of α–actinin-2 in tsA201 cells increased the measured I_Na densities as well as the Na_v1.5 protein at the cell surface. Colocalization of Na_v1.5 and α–actinin-2 was found mainly at the lateral membrane at the level of the Z-lines. α–actinin-2 seems to be an alternative to the dystrophin/syntrophin complex and ankyrin-g for the connection of Na_v1.5 to the cytoskeletal network. Whether these anchoring proteins have overlapping or clearly distinct functions remains to be investigated.

Synapse-associated Protein 97

Proteins of the membrane-associated guanylate kinase (MAGUK) family are expressed mainly at the cell-cell junctions and are characterized by numerous protein-protein interaction domains, including PDZ domains.⁸⁵ The MAGUK proteins regulate the function and localization of many membrane proteins (including ion channels) in neurons, epithelial cells, and cardiomyocytes. Synapse-associated protein 97 (SAP97) and zonula occludens 1 are the predominant MAGUK proteins expressed in cardiac cells.⁸⁶ SAP97 regulates the targeting, localization and function of cardiac K⁺ channels, such as Kir2.x,⁸⁷ K_v1.5,⁸⁸ and Kv4.x⁸⁹ via their PDZ domain–binding motifs located in the C-termini. We recently obtained evidence for the coexistence of at least two pools of Na_v1.5 channels in cardiomyocytes: one located at the lateral membrane with the dystrophin multiprotein complex, and the other with SAP97 at the intercalated discs (Figure 18-4).⁴ We demonstrated that the interaction of Na_v1.5 and SAP97 was dependent on the PDZ domain–binding motif that most likely interacts with one of the PDZ domains of SAP97. Reduction of the expression of SAP97 in both HEK293 and atrial cardiac cells led to a decreased I_Na without modification of any biophysical properties, suggesting that SAP97 may play a role in controlling the density of Na_v1.5 at the cell surface of cardiac cells. The detailed mechanisms underlying this regulation have not yet been investigated. SAP97 expression has been found at the intercalated discs^4,88 and the T-tubule compartment of cardiac cells.¹⁶ Milstein et al.¹⁶ recently demonstrated that Na_v1.5 expression is coregulated with the potassium channel K_ir2.1, and both interact with SAP97 (see Chapter 21 of this book).

Caveolin-3

Caveolae are plasma membrane invaginations with an enrichment of signaling molecules and ion channels.⁹⁰ Caveolin proteins are important constituents of these caveolae. Caveolin-3, encoded by the gene CAV3, is the predominant isoform expressed in striated muscle cells, including cardiomyocytes. Mutations in CAV3 have been linked to limb-girdle muscular dystrophy, rippling muscle disease, and familial hypertrophic cardiomyopathy.⁹¹ CAV3 has also recently been shown to be mutated in patients with congenital LQTS type 9⁹² and sudden infant death syndrome (SIDS).⁹³ Caveolin-3 was coimmunoprecipitated with Na_v1.5 in rat cardiac tissue⁹⁴ and HEK293 cells.⁹² The site of interaction between caveolin-3 and Na_v1.5 has not yet been determined (see Figure 18-1, B). Immunohistostaining of cardiac cells demonstrated that these two proteins are mainly colocalized at the lateral membrane.^92,94,95 Dystrophin is also a component of caveolae,⁹⁶ suggesting that the interaction between caveolin-3 and Na_v1.5 could be indirect via proteins of the dystrophin multiprotein complex.⁹ The precise role of the Na_v1.5/caveolin-3 interaction in normal physiology remains to be clarified. The coexpression of Na_v1.5 and the mutants of caveolin-3 found in patients with LQTS and SIDS in HEK293 cells was shown to increase the inward Na⁺-persistent current.^92,93 In an earlier study, it was reported that β-adrenergic stimulation by isoproterenol led to a rapid increase of peak I_Na in rat cardiac myocytes.⁹⁴ This phenomenon is most likely independent of protein kinase A, because a protein kinase A inhibitor did not reduce this effect. The increase was, however, completely abolished by antibodies against caveolin-3. The precise molecular and cellular mechanisms underlying these observations require further investigation.

α-1 Syntrophin and the Dystrophin/Utrophin Complex

Earlier work has shown that Na_v1.5 is part of the dystrophin multiprotein complex.⁹⁷ Our group demonstrated that Na_v1.5 interacts with dystrophin via adaptor syntrophin proteins.⁹ Similar to the binding with SAP97 and PTPH1, this interaction is dependent on the C-terminal PDZ domain–binding motif of Na_v1.5 (see Figure 18-1, A), which is composed of the last three residues of the protein. In the hearts of dystrophin-deficient mice (mdx), the best-studied animal model of Duchenne muscular dystrophy, the protein level of Na_v1.5 was decreased.⁹ The decreased expression of the Na⁺ channel resulted in reduced cellular I_Na and conduction defects, which were reflected on the electrocardiogram by a prolongation of the QRS complex duration, as well as conduction slowing in optical mapping experiments.⁴ The reduced expression of the Na_v1.5 protein could not be explained by a decrease in the SCN5A mRNA level,⁹ suggesting that a defect in the translational process or a lack of dystrophin may reduce the stability of the Na_v1.5 protein. Albesa et al.⁹⁸ showed that utrophin, a homologue protein of dystrophin, was upregulated in mdx hearts and interacted with Na_v1.5 similarly to dystrophin. Cardiac cells deficient in both dystrophin and utrophin displayed a larger decrease in Na_v1.5 expression and I_Na.⁹⁸ Two missense mutations in SNTA1, which encodes α1-syntrophin, have been described in patients with congenital LQTS,^99,100 further supporting the important role of this multiprotein complex in the regulation of Na_v1.5. The SNTA1 mutation, p.A390V,⁹⁹ was reported to disrupt a presently undescribed macromolecular complex comprising neuronal nitric oxide synthase, plasma membrane Ca-ATPase type 4b with syntrophin, and Na_v1.5. The overexpression of the mutant syntrophin protein in cardiac myocytes increased the persistent Na⁺ current, a finding consistent with the LQTS phenotype in affected individuals. The authors also described increased nitrosylation of Na_v1.5 when mutant syntrophin was coexpressed in HEK293 cells, suggesting that under basal conditions with wild-type syntrophin, Na_v1.5 nitrosylation is low because of the inhibition of neuronal nitric oxide synthase activity by Ca-ATPase. The syntrophin mutation may disrupt this complex and lead to increased nitrosylation of the channel, thus increasing the Na_v1.5-persistent current. Because syntrophin seems to be excluded from the intercalated discs,⁴ syntrophin-dependent regulation of the persistent current is most likely exclusively related to the lateral pool of Na_v1.5, suggesting that Na_v1.5-dependent late currents at the lateral membrane may be different from those at the intercalated discs.

Glycerol-3-Phosphate Dehydrogenase-like Protein

Mutations in the gene coding for Na_v1.5, SCN5A, are found in approximately 20% of patients with BrS. Other possible causative genes are still being investigated (see Chapter 92 ). London et al. described a locus on chromosome 3¹⁰¹ in a large family with BrS but simultaneously excluded SCN5A. A missense mutation of the gene coding for the glycerol-3-phosphate dehydrogenase-like protein (GPDlL) was later found.¹⁰² GPDlL is expressed in cardiac tissue and coexpression experiments using HEK293 cells showed that mutant GPDlL reduced the I_Na. Three more mutations of the GPDlL gene have been found in infants who died of SIDS.¹⁰³ Expression of these variants in neonatal mouse cardiomyocytes decreased I_Na, suggesting that a proportion of SIDS patients may have decreased I_Na, as is observed in BrS patients. Valdivia et al.¹⁰⁴ observed an interaction between Na_v1.5 and GPDlL by performing pull-down experiments. The site of interaction has not yet been investigated. The mechanisms by which the genetic variants of GPDlL reduce the Na⁺ current have been studied in expression systems.¹⁰⁴ A pathway has been proposed whereby Ser-1503 can be phosphorylated by protein kinase C (PKC) and leads to reduced I_Na. It has been shown that the activity of PKC depends on GPDlL function and that the mutant GPDlL variants lead to a further decrease of the I_Na. These observations linking the redox state of the cells with the activity of Na_v1.5 are very interesting and should be investigated using native cardiac cells and tissues.

Multicopy Suppressor of gsp1 (MOG1)

MOG1 is a small 29-kDa protein that is encoded by the RANGRF gene. MOG1 binds to the GTP-binding nuclear protein Ran and is involved in regulating nuclear protein trafficking.¹⁰⁵ MOG1 is found in the nucleus and the cytosol of cardiac cells, and its mRNA is well expressed in cardiac tissue.¹⁰⁵ Wu et al.¹⁰⁶ demonstrated that MOG1 interacts with the intracellular loop between domains II and III of Na_v1.5 (see Figure 18-1, A). This interaction was first described by performing a yeast two-hybrid screen, followed by pull-down and coimmunoprecipitation experiments.¹⁰⁶ The two proteins were also shown to colocalize in mouse ventricular cells, mostly at the intercalated discs. MOG1 coexpression in HEK293 cells increased the Na_v1.5-mediated current without altering its biophysical properties, suggesting that MOG1 is a cofactor for optimal channel expression at the cell membrane. The role of MOG1 in regulating the surface expression of Na_v1.5 was confirmed in a more recent study.¹⁰⁷ This study described two genetic variants of MOG1 found in patients with BrS that led to reduced expression of Na_v1.5 at the cell membrane of rat atrial cardiomyocytes and a decreased current. This work puts RANGRF on the increasing long list of susceptibility genes for BrS and illustrates the heterogeneity of mechanisms found in this syndrome.

Plakophilin2

Plakophilin2 is a desmosomal protein found in the intercalated discs of cardiac cells. The human gene (PKP2), which encodes plakophilin2, is mutated in patients with arrhythmogenic right ventricular cardiomyopathy.¹⁰⁸ By performing pull-down and coimmunoprecipitation experiments, Delmar’s group^19,20 demonstrated that Nav1.5 interacts not only with plakophilin2 but also with ankyrin-G and connexin43.²⁰ Whether the interactions between these different proteins of the intercalated discs are direct or indirect, as well as the site of interaction with Na_v1.5, remains to be determined. It was, however, convincingly shown that these proteins are colocalized at the intercalated discs, and that silencing of plakophilin2 in cardiomyocytes reduces the I_Na and alters some of its biophysical properties. The influence of plakophilin2 integrity on Na_v1.5 was demonstrated in vivo by studying a mouse model expressing only one allele of plakophilin2.²¹ Reduced expression of plakophilin2 decreased I_Na without changing the localization or total expression of Na_v1.5 in the mouse heart. These findings clearly support the notion of the coregulation of desmosomal proteins and the Na_v1.5 complex at the intercalated discs. The molecular and cellular mechanisms underlying these observations are still unclear.

Desmoglein2

The most recently described protein interacting with Na_v1.5 is desmoglein2, another protein of the desmosomal complex of cardiac cells found to be mutated in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC).¹⁰⁹ Desmoglein2 was shown²² to coimmunoprecipitate with Na_v1.5 in mouse cardiac tissue. The study of a transgenic mouse model overexpressing one mutant of desmoglein2 (p.N271S) revealed a reduction of the I_Na and electrical impulse propagation. There was, however, no reduction in Na_v1.5 total expression. These results are very similar to those seen with the plakophilin2 mutants (discussed earlier) and further support a cross-talk mechanism between the desmosomal proteins and Na_v1.5, whose molecular details remain to be investigated.

Ankyrin-G

The ankyrin proteins organize, transport, and anchor membrane proteins to the actin and spectrin cytoskeleton of cells.¹¹⁰ In the human genome, ANK1-3 are the three genes encoding ankyrins. The expression of ankyrin-B (ANK2) and ankyrin-G (ANK3) has been demonstrated in the heart.¹¹⁰ Mutations of ANK2 have been linked to congenital LQTS type 4,¹¹¹ sinoatrial node dysfunction, atrial fibrillation, conduction slowing, and sudden cardiac death, the collection of which define “ankyrin-B cardiac syndrome.”¹¹² There is currently no evidence that Na_v1.5 is directly regulated by ankyrin-B, even though in ankyrin-B–deficient mouse cardiac cells, Na⁺ channels display late openings similar to those seen in Na_v1.5 congenital LQTS type 3 mutant channels.¹¹³ On the other hand, ankyrin-G was shown to directly interact with the ankyrin-binding motif of the linker loop between domains II and III of Na_v1.5¹¹⁴ (see Figure 18-1, A, and Table 18-1). Ankyrin-G is predominantly located at the intercalated discs, where it interacts with not only Na_v1.5 but also the actin-associated protein βIV-spectrin and CaMKII.⁷⁰ A clear T-tubular localization of ankyrin-G has also been observed.⁷⁰ A SCN5A mutation, p.E1053K, in this motif was found in a BrS patient and was shown to disrupt the interaction between Na_v1.5 and ankyrin-G.¹⁵ Expression of wild type and mutant Na_v1.5 channels in adult rat myocytes¹⁵ was achieved using viral vectors, and although wild type Na_v1.5 channels were correctly transported to the intercalated discs and lateral membranes, the E1053K channels remained in the cytoplasm of the transduced cells.¹⁵ These findings were recently confirmed¹¹⁵ by silencing the expression of ankyrin-G in neonatal cardiomyocytes, which resulted in the reduction of Na_v1.5 expression and incorrect trafficking of most of the channels to the cell membrane.

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