Sodium Channels and Cardiac Excitation

In the heart, I_Na is the major current that excites cardiac cells in the atria, the ventricles, and the Purkinje fibers. Normally closed at the resting potential (approximately –85 mV in the adult working myocardium), sodium channels open upon depolarization beyond threshold, allowing an influx of Na⁺ ions down their electrochemical gradient. This inward current causes “all-or-none” membrane depolarization at a rapid rate (~500 V/s in the Purkinje fibers) in a process that moves the membrane potential to positive values. The rapid voltage-dependent activation of the sodium channel is immediately followed by an inactivation process that is also initiated by the initial depolarization. The inactivation process causes I_Na to be brief and results in the termination of the current. These properties are important for the rapid (≥1 m/s) conduction of the electrical impulse in the myocardium.

From the clinical standpoint, multiple mutations in the SCN5A gene coding for Na_V1.5 have been identified in association with the long QT syndrome (LQTS), Brugada syndrome, idiopathic ventricular fibrillation, cardiac conduction defects, and dilated cardiomyopathy associated with atrial fibrillation.⁷ Such mutations illustrate the pathophysiologic importance of these channels.

Homozygous knockout (KO) Scn5a^-/- mouse embryos die during mid-gestation, most likely because of severe defects in ventricular morphogenesis,⁸ which provides evidence for an essential role of Na_v1.5 in cardiac development.⁷ However, heterozygous KO mice (Scn5a^+/−) show normal survival but exhibit slow atrial, atrioventricular (AV) and intraventricular conduction, prolonged ventricular refractoriness, and enhanced ventricular arrhythmia inducibility.^7,8 Ventricular myocytes isolated from adult Scn5a^+/− mice demonstrate an approximately 50% reduction in sodium conductance.⁸ However, an important question that has never been addressed, is whether, as has been demonstrated in other model systems, changes in Na_v1.5 expression alter the level of functional expression of other membrane ion channels, particularly Kir2.1.⁵ Based on recent results,⁵ it would be reasonable to expect a significant reduction in Kir2.1 (and its respective ionic current, I_K1) and in the RMP of adult ventricular cardiomyocytes as a consequence of one SCN5A allele being absent. Should that be the case, one would predict that I_K1, the RMP and excitability would be rescued to normal levels by virally mediated gene transfer of Na_v1.5 into adult cardiomyocytes isolated from these mice.

In a recent study using a transgenic mouse line overexpressing the human SCN5A gene,⁹ levels for the cardiac sodium mRNA transcript (Scn5a) and protein (Na_v1.5) were each increased by approximately tenfold. However, no abnormalities were found in the electrophysiology of the ventricles. The QRS duration and the corrected QT interval (QTc) in SCN5A overexpressing mice were indistinguishable from their nontransgenic littermates. In addition, no ventricular arrhythmias were detected in the transgenic mice.⁹ The sodium current densities and APDs from transgenic ventricular cardiomyocytes were nearly identical to those of nontransgenic cells. Equally striking were the similarities between transgenic and normal hearts with respect to the sodium current densities and APDs found in atrial cells. However, baseline ECG recordings by telemetry revealed a shortened PR interval and P wave duration in the transgenic mice compared to their littermate controls, which the authors interpreted as being the result of altered AV nodal conduction.⁹ However, the possibility that His-Purkinje conduction was enhanced and thus caused the PR shortening was not considered. Nevertheless, it was clear that overexpression of SCN5A did not significantly increase the surface density of sodium channels in ventricular or atrial myocytes.⁹ This finding is different from what was found in the cultured neonatal rat myocyte. In these cells, I_Na is inherently relatively small, but viral transfer (and therefore overexpression) of Na_v1.5 is accompanied by a significant increase in Na_v1.5 and I_Na, excitability, and CV. As suggested by Abriel,⁷ one possible explanation to account for the disparate results of the two studies may be that the sodium channels are binding in a macromolecular complex. Based on this premise, and the assumption that there are a limited number of docking sites or complexes to which the sodium channel binds in the adult heart, then this complex (i.e., including its localization, stoichiometry, available components) could provide an upper limit as to how many functional sodium channels can be brought to the membrane in the adult mouse myocyte.⁷ However, such an idea might not be tenable in light of recent experiments demonstrating that transgenic overexpression of Kir2.1 increases I_Na density in the adult mouse heart (discussed later).

The Inward Rectifying Potassium Current (I_K1)

Among the three strong inward-rectifying potassium channels (Kir2.1, 2.2 and 2.3) that are expressed in the heart, Kir2.1 is the most abundant in the ventricles. Kir channels are responsible for I_K1, and they are involved in the depolarization, repolarization, and the resting phases of the cardiac action potential.10,11 It is usually accepted that, near the resting potential, the ventricular I_K1 conductance is much larger than that of any other current, with the exception of the adenosine triphosphate (ATP)-sensitive potassium current (I_KATP), which, however, is generally not present since the KATP channels are not open under normal conditions. It is thus likely that physiological and/or pathophysiological modulation of I_K1 will have a significant effect on excitability. The Kir channels show strong rectification between –50 and 0 mV, which means that they remain closed during the AP plateau; they only open when the membrane potential repolarizes to levels between –30 and –80 mV, which occurs during the late phases of the action potential.¹² Rectification is achieved by a voltage-dependent blockade by intracellular magnesium and/or one of the polyamines (putrescine, spermine, spermidine),¹² which are known to interact with at least three amino acid residues located inside the pore of the channel complex.¹³ Investigators have used several strategies to modify and study Kir2.1, including a knockout mouse,¹⁴ antisense oligonucleotide targeting,¹⁵ and DNA transfection of a dominant-negative construct.¹⁶ These studies have helped to define the role of I_K1 in cardiac excitability.^17,18 As shown by Zartizky et al., ventricular myocytes from Kir2.1 knockout (Kir2.1^−/−) mice lack I_K1 in whole-cell recordings under physiologic conditions, which demonstrated that Kir2.1 is the major determinant of I_K1.¹⁴ In that model, sustained outward K⁺ currents and Ba²⁺ currents through L- and T-type channels were not significantly altered by the mutation. However, the direct consequences of Kir2.1 disruption on Na_v1.5 function were never studied in the homozygous Kir2.1 KO mice. Recently, the authors took advantage of the availability of the heterozygous Kir2.1 KO (Kir2.1^−/+) mouse model to study the functional consequences of reducing Kir2.1, and therefore I_K1, at both cellular and organismal levels. In particular, as discussed in detail below, the Kir2.1^−/+ mouse was used to address the question of whether reduced Kir2.1 protein is associated with reduced expression of Na_v1.5.⁵

Loss of function mutations in the KCNJ2 gene, which codes for Kir2.1, have been identified in patients affected by Andersen-Tawil syndrome (ATS), also known as LQTS type 7, which is characterized by prolonged repolarization.¹⁹ In addition to being expressed in the heart, Kir2.1 is expressed in other organs, such as skeletal muscle. As a result, ATS is associated with hypokalemic periodic paralysis and skeletal developmental abnormalities, including clinodactyly, low-set ears, mandibular hypoplasia, short stature, and scoliosis.¹⁹ In the heart, reduction of I_K1 leads to QT prolongation and predisposes to arrhythmias; however, QT prolongation is less prominent in patients with ATS than in those with other types of LQTS.²⁰ Moreover, although ATS patients develop ventricular tachyarrhythmias, including torsades de pointes, sudden cardiac death is rare.²⁰

In patients with short QT syndrome type 3, a gain-of-function mutation (D172N) in the KCNJ2 gene was demonstrated.²¹ The D172N mutation causes a significant increase in the outward component of the I-V relation of I_K1, and it is associated with an accelerated repolarization, which can be arrhythmogenic. However, the direct involvement of I_K1 in arrhythmia mechanisms was not demonstrated in the affected patients; therefore, the mouse model of I_K1 overexpression²² was used to study the effect of I_K1 increase on VF at the molecular level.²³ An increase of I_K1 was shown to shorten repolarization and the QT interval and to exert a proarrhythmic effect in both the atria and the ventricles of this transgenic mouse model.^22,23 Optical mapping and numerical studies in these mice demonstrated that, by increasing the RMP, I_K1 overexpression enhances the availability of sodium channels during sustained reentry, which contributed to the observed increase in the frequency and stability of rotors and ventricular fibrillation.²³ Recent experiments with the same Kir2.1 overexpressing mouse model have shown that Kir2.1, and therefore I_K1, upregulation leads to a significant increase in the density of I_Na. These results strongly support the hypothesis that a change in the functional expression of Na_v1.5 could be the result of protein-protein–type interactions with Kir2.1. Such interactions can be mediated through common partners in a macromolecular protein complex.⁵

Ankyrin-G and Na_v1.5 Trafficking

A number of accessory proteins have been shown to interact and form a multiprotein complex with Na_v1.5.7,47 Ankyrin-G and Na_v1.5 are both localized at the intercalated disc and at T-tubule membranes in cardiomyocytes, and Na_v1.5 coimmunoprecipitates with the 190-kDa ankyrin-G in detergent-soluble lysates from rat heart.⁴⁷ The two proteins interact through a 9-amino acid motif in the Na_v1.5 cytoplasmic loop II between DII and DIII, which helps to promote the localization of sodium channels to the cell membrane in cardiomyocytes.⁴⁷ In 2004, Mohler et al.⁴⁷ identified a point mutation (E1053K) in the ankyrin-binding motif of Na_v1.5 that is associated with Brugada syndrome. The E1053K mutation abolishes binding of Na_v1.5 to ankyrin-G and prevents the accumulation of Na_v1.5 at cell surface sites in ventricular cardiomyocytes.⁴⁷ Those data suggested that association of Na_v1.5 with ankyrin-G is required for Na_v1.5 localization, and therefore function, at excitable membranes in cardiomyocytes. Concerted ankyrin-G interaction with potassium (Kv7) and Na_v channels has also been demonstrated in neurons,⁴⁸ but Kir2.1 has not been shown to interact with ankyrin-G. There is still the possibility that a defect in ankyrin-mediated Na_v1.5 trafficking and localization on the membrane could alter Kir2.1 functional expression. This idea has not yet been explored in any detail.

Na_v1.5 Is Regulated by SAP97

The C-terminus of Na_v1.5 contains 244 amino acid residues that have been shown to interact with a number of proteins, including fibroblast growth factor-homologous factor 1B, calmodulin, Nedd4-like ubiquitin-protein ligase, dystrophin and syntrophin, Fyn, and PTPH1 (protein-tyrosine phosphatase).49–51 Of importance, the three last three amino-acids (Ser-Ilc-Val, SIV) of the Na_v1.5 C-terminus constitute a PDZ-domain binding motif that is known to interact with PDZ domains found in proteins of the membrane-associated guanylate kinase (MAGUK) family.⁵² MAGUK proteins act as scaffolding proteins involved in intermolecular interactions and protein trafficking to the cell membrane. Petitprez et al.⁵³ reported their study on the interaction between SAP97, one of the cardiac MAGUK proteins, and Na_v1.5, with the idea of demonstrating that the Na_v1.5-SAP97 interaction is involved in the turnover and regulation of the biophysical properties of Na_v1.5. Using Na_v1.5 C-terminal fusion proteins in pull-down experiments with human and mouse heart protein extracts, these investigators demonstrated that the association between SAP97 and Na_v1.5 depended on the PDZ-domain binding motif of Na_v1.5. The interaction appeared to be specific for SAP97 and Na_v1.5, because the Na_v1.5 constructs did not pull down either PSD95 or ZO-1, two other MAGUK proteins that are expressed in the human heart.⁵⁴ In patch-clamp experiments, Petitprez et al.⁵³ further demonstrated that silencing SAP97 expression reduced the whole-cell sodium current without decreasing the total protein content in HEK293 cells stably expressing Na_v1.5 channels. Taken together, with the demonstrated colocalization of Na_V1.5 and SAP97 at both intercalated discs⁵³ and T-tubules,⁵ these findings support the existence of an interaction between Na_v1.5 and SAP97 in cardiac tissue. This interaction could have a role in determining the channel density at the plasma membrane. Therefore, Petitprez et al.⁵³ have suggested that there are at least two pools of Na_v1.5 channels in cardiomyocytes: one targeted to lateral membranes by the syntrophin-dystrophin complex, and another targeted to the intercalated discs by SAP97.

Na_v1.5 Interacts with Syntrophins