Ca2+-dependent modulation of voltage-gated myocyte sodium channels

Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III–IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.


Introduction
Transmembrane action potential initiation and propagation, mediated by surface membrane Na + channel (Nav) proteins, is strategic to activation in excitable cells, of which skeletal and cardiac myocytes constitute important examples. The activation process feeds forward into a ryanodine receptor (RyR) mediated release of sarcoplasmic reticular (SR) store Ca 2+ . The consequent elevation of cytosolic Ca 2+ concentration [Ca 2+ ] i is central to initiation of myocyte contraction. Ca 2+ is additionally a strategic second messenger with signalling actions regulating protein activity through widespread cell types. This article addresses recent interest in possible Ca 2+ feedback signalling on the Na + channel itself, its possible physiological significance, and implications for human disease in skeletal and cardiac muscle. We relate the voltage sensing, and channel opening and inactivation processes in skeletal, Nav1.4 and cardiac Nav1.5 to their potential regulation at direct and indirect Ca 2+ binding and phosphorylation sites. This includes its III-IV linker region and its interactions with its C-terminal domain, whose different regions are associated with widespread mutations related to skeletal and cardiac muscle disease. We examine in vitro studies in expression systems exploring for direct and indirect effects of Ca 2+ on channel properties, then extend these to physiological studies in both skeletal and cardiac myocytes in situ, from experimental platforms using normal hearts, and those modelling genetic Ca 2+ homeostatic disease, broadening these to genetic exemplars for more common human disease types. inactivated state [2][3][4][5][6]. Indeed, these structures represent the IFM motif, as expected, engaged with an allosteric intracellular DIII site. In addition however, two separate, short α-helical regions of the DIII-DIV intracellular linker, site A and site B (equivalent to helix 0 of DIV: Figure 1A), make contacts with intracellular sites on DIV, probably further stabilizing the inactivated state (Figure 2A,B) [6]. However, if the engagement of the DIII-DIV linker and IFM motifs with these allosteric sites is indeed critical for promoting the inactivated state, then they must adopt different conformations in the resting and open states.
Nav channels also include a regulatory, globular, intracellular C-terminal domain (CTD), highly conserved amongst Nav subtypes ( Figure 3A), connected to the DIV S6 helix via a flexible and disordered linker ( Figure 1A). The CTD begins from amino acids 1599 in Nav1. 4 and 1773 in Nav1.5, with a sequence of five α-helical regions fitting the consensus sequence for an EF-like hand (EFL) ( Figure 3A,B) [7], with the latter part a fibroblast growth factor (FGF) homologous factor (FHF) binding site. It is followed by a sixth α-helical region ( Figure 3A,B) and ends with a more disordered and less-well structurally characterized region containing short motifs likely controlling cytoskeletal binding and ubiquitination [8], including a Nedd4-like binding domain, PY motif domain and a syntrophin-anchoring PDZ binding motif ( Figure 3A) [9].
NMR analysis of purified EFLs indicates the presence of a prominent cleft in the EFL, bounded by α-helices [10]. This cleft can complex with Site A of the DIII-DIV linker (Figure 2A,B). Modelling of dynamic interactions between the DIII-DIV linker and the CTD through the Nav channel cycle in mammalian Nav1.7 and cockroach NavPas channel structures [11] suggested that in the channel closed state, acidic residues on the CTD EFL domain form salt bridges with basic residues on the DIV S4 helix, whilst Site A of the DIII-DIV linker is held in (A) Sequence alignment of the DIII-DIV linkers from Nav1.5 and Nav1.4. Identical residues indicated by (*), conservative changes by (:) and semi-conservative changes by (.) below the sequence alignments. IFM motifs indicated in red. Site A and site B helices boxed. In the Nav1.5 sequence, examples of residues whose mutations are associated with Long QT syndrome (LQT3) indicated by ( †) and with Brugada syndrome (BrS) by (#). LQT3 and BrS-associated residues implicated in binding of the DIII-DIV linker to the α-subunit and to Ca 2+ -calmodulin coloured orange and sky blue, respectively. In the Nav1.4 sequence, residues whose mutations are associated with myotonia indicated by (|) and with paramyotonia the CTD EFL cleft. As a consequence, the IFM motif is physically constrained and prevented from prematurely engaging with the inactivated state [11,12]. Upward movement of the DIV S4 helix accompanying channel opening, disrupts these salt bridges. CTD dissociation from the DIII-DIV linker then frees the IFM motif permitting transition into the inactivated state ( Figure 4A). Most of the cryo-EM structures thus do not show a resolved CTD [2][3][4][5]. This suggests that in the inactivated state, the CTD is free to adopt multiple conformations with respect to the bulk of the Nav channel, constrained only by its tethering to the S6 helix [11].

Intracellular Ca 2+ as potential Nav modulator
In skeletal and cardiac muscle, the RyR-mediated SR Ca 2+ release following Nav1.4 or Nav1.5-mediated depolarization elevates bulk [Ca 2+ ] i from ∼100 nM to 1-10 mM causing contractile activation. In addition, recent reports implicated cytosolic Ca 2+ in a feedback Nav modulation whether through Ca 2+ by itself or following its binding to the modulator protein calmodulin [8,13]. The latter nM-low mM K d Ca 2+ sensor contains N-and C-lobes, each possessing two Ca 2+ -binding EF hands. In turn, Ca 2+ [14][15][16]. (A) Sequence alignment of CTDs from Nav1.5 and Nav1.4. Identical residues are indicated below the sequence alignment by (*), conservative changes by (:) and semi-conservative changes by (.). Locations of the linker region, EF hand and helix 6 highlighted. The extended region of helix 6 containing sequences implicated in apo-or Ca 2+ -calmodulin binding coloured cyan. Within this region, the consensus IQ-motif is indicated. In the Nav1.5 sequence, examples of LQT3 and BrS-associated residues coloured orange and sky blue, respectively. In the Nav1.4 sequence, myotonia and PMC-associated residues coloured tan and purple, respectively. (B) Comparative structures of CTDs from Nav1.5 (a-c) and Nav1.4 (d,e) with apo-calmodulin (a,d) or Ca 2+ -calmodulin (b,c,e), in side view and top view. To emphasize the variety of ways in which calmodulin can bind to helix 6, the EF hands have been removed from the top views and the orientation of each helix 6 structure has been arbitrarily standardized, with Nav1.5 residue R1897 and its Nav1.4 equivalent K1723 placed at 12 o'clock. In the Nav1.5 structures, LQT3 and BrS-associated residues highlighted as spheres and coloured orange and sky blue, respectively. In the Nav1.4 structures, myotonia and PMC-associated residues highlighted as spheres and coloured tan and purple, respectively. Ca 2+ ions shown as red balls.
Biophysical studies on isolated protein fragments demonstrate that Site A and Site B of the Nav DIII-DIV linker bind to the C-and N-lobes of Ca 2+ -calmodulin, respectively. But this interaction does not occur with the C-or N-lobes of apo-calmodulin (Figure 2A,C) [17,18]. As noted above, Site A can also bind the CTD EFL cleft. Interestingly, Nav1.5 DIII-DIV linker and CTD co-precipitation occurs in the presence of Ca 2+ -calmodulin, but is inhibited by the Ca 2+ -chelator, EGTA. This could indicate that Ca 2+ -calmodulin acts catalytically to load the DIII-DIV linker onto the CTD [19] (see below).
It had previously been suggested that Nav1.5 CTD EFLs could bind Ca 2+ directly [7,9,13]. However, in Ca 2+ -binding EF hands, such as those occurring in calmodulin, the Ca 2+ -chelating acidic residues typically lie within turn loops between adjacent α-helices. This pattern is not seen in the CTD-EFL domain [10,20,21]. On the other hand, the CTD, with its significant homologies between Nav subtypes, illustrated for Nav1.4 and Nav1.5 ( Figure 3A), binds calmodulin. So, this is the most likely mechanism by which the CTD senses [Ca 2+ ] i . The IQ motif within helix 6 of the Nav1.5 CTD [22] ( Figure 3A) can bind the apo-calmodulin C-lobe [14]. Additionally, both the EFL domain and the N-terminal of helix 6 can bind the apo-calmodulin N-lobe ( Figure 3Ba). Following Ca 2+ -calmodulin binding, the IQ motif ( Figure 3A) can bind the 'semi-open state' Ca 2+ -calmodulin C-lobe. But now a downstream, slightly overlapping N-lobe binding motif (NLBM) ( Figure 3A) can bind a shifted Ca 2+ -calmodulin N-lobe (Figure 3Ba,b). An alternative structure (PDB: 6mud) for the Nav1.5 CTD Ca 2+ -calmodulin complex is shown in Figure 3Bc [23]. Here, the Ca 2+ -calmodulin N-lobe is untethered to the CTD, and the Ca 2+ -calmodulin C-lobe adopts a strikingly different orientation on helix 6 ( Figure 3Bc). However, the CTD construct used in this structure contained a truncated NLBM motif, so that its binding to Ca 2+ -calmodulin N-lobe was likely compromised [23]. Interestingly, a BrS mutation A1924T [24] ( Table 1) occurs within the Nav1.5 NLBM site, suggesting that the structure shown in Figure 3Bc could represent an abortive complex, leading to a BrS phenotype. Nav1.4 lacks a functioning NLBM ( Figure 3A), whence  linker fully engaged with the α-subunit and the CTD dissociated from site A. Ca 2+ levels are assumed to be elevated following opening of voltage-gated Ca 2+ channels, so that Ca 2+ -calmodulin binds to helix 6 (PDB structure 4jq0). (b-e) Possible, sequential conformational changes occurring during the recovery from inactivation steps (see text for details). (f ) Proposed Nav1.5 conformation after return to the closed (resting) state. Since Ca 2+ levels are now low, apo-calmodulin binds to helix 6 (PDB structure 4ovn). Ca 2+ shown as red balls.  [23] and it is striking that the rearrangements of calmodulin on the Nav1.4 CTD helix 6 are noticeably less pronounced compared with Nav1.5 (Figure 3Bd,e)) The CTD and DIII-DIV linker of both Nav1.4 or Nav1.5 show mutations associated with specific disease phenotypes. These respectively involve skeletal or cardiac muscle electrophysiological function (Table 1) [25]. Interestingly, within the DIII-DIV linker, gain of Nav1.5 function LQT3 mutations cluster in Site A and affect residues that stabilize DIII-DIV linker binding to the intracellular face of DIV (Figure 2A,B) [26]. In the CTD, the LQT3 mutants tend to occur on helix 6, within and around the IQ motif anchoring apo-calmodulin, as well as contact sufaces between helix 6 and the EFL domain [26] ( Figure 3A). These mutations are rescued by overexpressed calmodulin [27].
Contrastingly, loss of Nav1.5 function, Brugada Syndrome (BrS), mutations mainly occur in Site B of the DIII-DIV linker [26] (Figure 2A). One exception, however, is Site A residue Y1494. Mutations in this residue are associated with BrS, not LQT3 ( Figure 2A). It may be significant that in the presumed inactivated state structure, residue Y1494 points away from the inactivation site on the intracellular region of DIV ( Figure 2B), but in the Ca 2+ -calmodulin C-lobe/Site A complex, it now lies within the protein docking site ( Figure 2C) [17]. Thus, BrS and LQT3-associated mutations in Site A, may perturb different molecular contacts. In the CTD, residues associated with BrS cluster particularly within the EFL cleft ( Figure 3A,B). This could compromise the capture of the DIII-DIV linker and compromise recovery from inactivation ( Figure 4A). In Nav1.4, mutations in two EFL residues, Q1633 and F1705 are associated with myotonia and paramyotonia congenita (PMC), respectively ( Figure 3A). In the Nav1.4 EFL structure, these two residues lie suggestively close to each other, where they could help stabilize the EFL cleft (Figure 3Bd,e).
In summary: site A of the DIII-DIV linker can bind to an intracellular site on Nav α-subunit DIV, when the channel is in the inactivated state ( Figure 2B). Yet it can also bind to the Ca 2+ -calmodulin C-lobe ( Figure 2C) and to the CTD-EFL domain, when the channel is in the closed state [11]. Similarly, site B of the DIII-DIV linker can bind to DIV on the inactivated Nav α-subunit ( Figure 2B), but also to the Ca 2+ -calmodulin N-lobe ( Figure 2C). Furthermore, in several cases, the same amino acid residues contribute to the different binding states ( Figure 2B,C). Thus, within a given channel, these interaction states must be mutually exclusive. Finally, as noted above, the cryo-EM structure ( Figure 1B), suggests that the CTD does not bind the DIII-DIV linker when the channel is in the inactivated state [6]. The simplest interpretation is that these different binding states can only take place at specific points during the activation/inactivation/recovery from inactivation cycle of the channel and thus could help impose directionality onto the process.
This idea is outlined in schematic form for the whole Nav activity cycle in Figure 4A and for the role of calmodulin in the recovery from inactivation steps in Figure 4B. One may suggest that immediately after Nav1.5 inactivation, Site A and B, and the IFM motif of the DIII-DIV linker, are all fully engaged with their sites on the α-subunit DIII, and the CTD does not bind the DIII-DIV linker (Figure 4Ba). With an elevated [Ca 2+ ] i , the interaction between Ca 2+ -calmodulin and the CTD is represented by structure PDB: 4jq0 ( Figure 3Bb). As the membrane potential hyperpolarizes, the voltage sensing helices of DIII and DIV return to their resting states. Site A and the IFM motif detach from their sites on DIV (Figure 4Bb). The Ca 2+ -calmodulin C-lobe can then bind Site A, adopting the conformation shown in PDB: 4djc ( Figure 2C, upper panel). Further rearrangements allow the Ca 2+ -calmodulin N-lobe to bind to Site B as in PDB: 5dbr ( Figure 2C lower panel). Together, this could act like a ratchet to prevent the reattachment of Sites A and B and thus the IFM motif to DIV (Figure 4Bc) [18]. There must be further rearrangements to free the calmodulin C-lobe from Site A and the calmodulin N-lobe from site B, so that Site A can reattach to the cleft in the EFL domain of the CTD (Figure 4Bd-f ) [21]. Since the affinity of calmodulin for Site A and B is strictly Ca 2+ -dependent, [18], this could take place as [Ca 2+ ] i returns to its resting state, (Figure 4Bf ).
Other Nav sites may potentially be involved in Ca 2+ -mediated regulation. Thus, CaMKII-mediated phosphorylation of particular (Ser516, Ser571, and Thr594) residues within the DI-DII intracellular linker region increases late I Na delaying action potential repolarization, characteristic of LQT3 [28]. However, an existence of calmodulin-KN93 interactions could result in attribution of modified protein function to CaMKII phosphorylation rather than calmodulin action. KN93 may also impair calmodulin-III-IV linker domain interaction and I Na recovery from inactivation [29]. Phosphorylation at a protein kinase C specific site reduced peak I Na and shifted (by −15 mV) steady state inactivation V 1/2 [30]. Mutations at a Nav1.5 N-terminal domain calmodulin binding site down-regulated I Na [31]. Elevated [Ca 2+ ] may also up-regulate Nedd4-2 in turn targeting Nav1.5 for degradation via a CTD PY motif [32].
Ca 2+ uncaging investigations extending to skeletal myotubes derived from mouse glt cells similarly demonstrated Ca 2+ -mediated Nav1.4 regulation at sensitivities appropriate for physiological Ca 2+ transients, but no such Nav1.5 regulation in adult guinea-pig ventricular myocytes [36]. However, in freshly isolated rabbit ventricular myocytes, [Ca 2+ ] i elevations produced by Ca 2+ -BAPTA (0-500 nM)-buffered patch-clamp electrode solutions or caffeine challenge caused parallel reductions in I Na density, unit channel amplitudes and maximum action potential upstroke rates (dV/dt) max , without altering steady state voltage dependences of I Na activation or inactivation [40]. Cultured rat neonatal ventricular cardiomyocytes also showed altered Nav expression with more sustained alterations in intracellular Ca 2+ homeostasis. Nav1.5 mRNA levels then altered in parallel with decreases or increases in whole cell patch-clamp I Na with 24 h sustained elevations (10 mM) or Figure 6. Na + current down-regulation in native murine skeletal muscle fibres by altered Ca 2+ homeostasis following caffeine induced ryanodine receptor (RyR) activation, abrogated by dantrolene mediated RyR antagonism.
(A)(a) Double pulse protocol from a hyperpolarized prepulse potential V 0 to activating voltage V 1 followed by further depolarization to fixed depolarized voltage V 2 , respectively assessing (b) Na + current activation and subsequent inactivation produced by the voltage step to V 1 . (B) 2-10 mM caffeine increases integrated background aequorin Ca 2+ signal (upper trace) and twitch force (lower trace) in rat fast twitch muscle at 25°C over timecourses dependent upon caffeine concentration. Arrows denote periods of caffeine exposure. (C) Families of loose-patch clamp membrane currents in response to the double pulse protocol before (a, b) and at successive intervals ((i)-(iv)) following introduction (c, d) of caffeine (0.5 mM) before (a, c) and following (b, d) addition of dantrolene (10 mM). Currents expressed as current densities ( pA/mm 2 ) through 28-32 mm

BAPTA-AM-mediated reductions of culture media [Ca 2+
]. These also occurred without alterations in single conductance, or activation and inactivation properties [41].
These varied observations could arise from a range of possible Nav Ca 2+ sensing mechanisms, including direct Ca 2+ binding to the first EF-like hand [7,15,35], or Ca 2+ -calmodulin or apo-calmodulin binding to, the CTD [34,36]. The latter possibilities were compatible with reported calmodulin binding to peptide channel fragments [42,43]. Finally, structural studies invoked possible Nav regulatory sites alternative to the CTD including the III-IV loop [17]. At all events, this available evidence permits a direct in vivo regulation of Nav-mediated excitable activity by intracellular Ca 2+ , involving mechanisms highly conserved among all nine Nav isoforms. This could complement or replace hypotheses invoking [Ca 2+ ] i -mediated increases in electrogenic Na + /Ca 2+ exchanger (NCX) activity in cardiac muscle under pro-arrhythmic conditions [44]. The latter may mediate delayed after depolarization (DAD) phenomena and is also implicated in altering action potential recovery as opposed to initiation and propagation activity. NCX may also increase [Na + ] i thereby influencing transmembrane Na + electrochemical gradients. However, this would involve mM-levels corresponding to the altered [Ca 2+ ] i as opposed to normal background nM-[Na + ] i levels. Furthermore, NCX activity is not a prominent normal skeletal as opposed to cardiac muscle feature. Nevertheless, in either event, over the long term, reduced or increased background [Ca 2+ ] i resulting from sustained low or high firing rates could furnish a form investigating Na + channel activation and inactivation as in Figure 5A. Na + currents in response to (B) activation by depolarization to level V 1 and (C) following their inactivation, to final level V 2 following their inactivation at level V 1  of Ca 2+ memory modifying Nav expression or gating and therefore its availability for driving action potential upstroke and propagation. In skeletal muscle, this could reduce cell excitability permitting recovery from fatiguing stimulation. However, the accompanying conduction velocity (CV) reductions could contribute to pathological cardiac arrhythmic or epileptiform nerve cell phenotypes.
Native skeletal and cardiac myocytes show acute Ca 2+ -dependent I Na modulation In vivo Ca 2+ -dependent Nav modulation was observed in native cardiac or skeletal myocytes in intact physiological systems and clinical disease models. Use of loose, as opposed to conventional cell-attached, patch-clamp methods, avoided Ca 2+ perturbations produced by the measurement method itself. I Na families recorded from voltage steps from resting to sequentially depolarized activating test potentials, followed by further pulses to a fixed depolarized level to evaluate the resulting channel inactivation (Figure 6Aa,b) were compared before and following perturbations of their in vivo Ca 2+ homeostatic mechanisms. Studies in both skeletal and cardiac myocytes demonstrated potentially physiologically significant negative feedback regulation of Nav1.4 and Nav1.5 by RyR-mediated release of intracellularly stored SR Ca 2+ . In murine skeletal muscle, acute RyR2 activation by the exchange protein directly activated by cAMP (Epac) by the activator 8-(4-chlorophenylthio)adenosine-3 0 ,5 0 -cyclic monophosphate (8-CPT, 1 mM) [45], reduced maximum I Na whilst leaving V 1/2 values unchanged, actions abrogated by the RyR-inhibitor dantrolene (10 mM) [46]. The RyR agonist caffeine, at concentrations of 0.5 or 2 mM, produced sustained activation or transient activation followed by inactivation, of RyR-mediated SR Ca 2+ release and corresponding parallel alterations in [Ca 2+ ] i [47; Figure 6B]. These changes directly paralleled time-dependent decreases or increases in peak I Na values (Figure 6Cc,d) also abrogated by dantrolene (10 mM). Finally, dantrolene applied by itself produced small increases in I Na , suggesting inhibitory effects of even background Ca 2+ release on I Na ( (Figure 6Ca,b) [48], potentially through formation of microdomains localizing [Ca 2+ ] i heterogeneities in junctional regions separating the T-tubular and SR membranes [49] Elevating [Ca 2+ ] i by applications of high extracellular [Ca 2+ ], caffeine, and the SR Ca 2+ ATPase inhibitor cyclopiazonic acid in murine atria [50], in addition to 8-CPT in murine atria and ventricles, all reduced mean peak inward I Na . 8-CPT (1 mM) induced Ca 2+ homeostatic changes manifesting as spectrofluometrically measured spontaneous Ca 2+ waves in murine atrial myocytes ( Figure 7A) [45]. These findings accompanied 30-50% reductions in inward I Na ( Figure 7B,C), abrogated by dantrolene (10 mM), which by itself left I Na at pre-treatment levels. Inactivation V 1/2 and k ( Figure 7D), and time constants for Na + current recovery from inactivation remained unchanged [51]. Intracellular sharp microelectrode membrane potential recordings in intact Langendorff-perfused preparations correspondingly demonstrated reduced maximum atrial and ventricular (dV/dt) max [51]. Action potential latencies reflecting delayed conduction increased while action potential durations and refractory periods were unchanged. The hearts also showed increased ventricular arrhythmic incidences following rapid pacing or extrasystolic stimuli [52].
Ca 2+ -dependent I Na modulation may underly skeletal muscle cold-aggravated myotonia A first clinical example of a C-terminal Nav1.4, SCN4A, mutation associated with human disease is cold-aggravated myotonia, which causes transient myotonic stiffness or renders fibres transiently inexcitable resulting in a periodic paralysis ( Table 1). The SCN4A mutant concerned contained two predicted amino acid substitutions, a DIS5-S6 loop T323M and an intracellular C-terminus F1705I substitution. Whole cell patch clamp I Na from transiently transfected HEK293 cells expressing Nav1.4-T323M were indistinguishable from WT, consistent with a benign polymorphism. However, Nav1.4-F1705I channels showed a slowed fast inactivation with a positive 8.6 mV shift in steady-state voltage-dependence often associated with myotonia, but normal activation, recovery from fast inactivation or persistent current [53].
CPVT is also associated with atrial fibrillation similarly attributed to abnormal Ca 2+ homeostasis particularly following increased sympathetic tone [58]. In superfused RyR2-P2328S/P2328S atrial preparations, loose patch clamp measurements also demonstrated reduced peak I Na with otherwise normal activation and inactivation current-voltage relationships ( Figure 8A,Ba) [50]. Floating intracellular microelectrode measurements demonstrated reduced (dV/dt) max and interatrial CVs though normal action potential duration amplitudes and refractory periods (Figure 8Bb,c) while multi-electrode arrays detected reduced atrial epicardial action potential CVs in RyR2-P2328S/P2328S atria when compared with WT [59]. Intrinsically active and regularly stimulated RyR2-P2328S/P2328S but not wild-type atria correspondingly showed frequent sustained tachyarrhythmias, delayed afterdepolarizations and ectopic action potentials. Extrasystolic S2 stimulation provoked arrhythmia at longer S1S2 intervals in RyR2-P2328S/P2328S than WT atria, nevertheless corresponding to similar (dV/dt) max , and effective interatrial CVs as in WT [59]. Gain-of-function skeletal muscle RYR1 mutations are associated with a malignant hyperthermia typically following halothane anaesthesia. Reports of increased slowly inactivating inward, tetrodotoxin sensitive current in cultured human malignant hyperthermia skeletal myocytes may prompt further investigations into possible electrophysiological, Nav1.4 phenotypes [60]. Anti-arrhythmic targeting of Ca 2+ homeostasis in clinical CPVT, cardiac failure and hypertrophic cardiomyopathies The above properties may underpin reported paradoxical pro-and anti-arrhythmic actions of low (1 mM) flecainide concentrations in WT and RyR2-P2328S/P2328S murine atria. Flecainide blocks both Nav1.5 and RyR2 with IC 50 s of 2-7 mM and 5-11 mM, respectively [61][62][63]. Either effect could potentially rescue an elevated [Ca 2+ ] i . On the one hand, flecainide's Class Ic Nav1.5 blocking action causes a pro-arrhythmic CV slowing; however, action of a consequently reduced [Na + ] i on NCX could reduce pro-arrhythmic [Ca 2+ ] i elevations [64][65][66]. In intact WT hearts, flecainide (1 mM) exerted atrial pro-arrhythmic effects, accompanying reduced loose patch clamp I Na and multi-electrode array recorded CV, whilst sparing refractory periods (Figure 9Aa,Ba,Ca). On the other hand, in RyR2-P2328S/P2328S atria, flecainide paradoxically rescued increased arrhythmic frequency. However, in contrast with its Nav1.5 inhibitory action in WT, it rescued I Na and maintained CV at WT values, leaving refractory periods unchanged (Figure 9Ab,Bb,Cb), effects directly replicated by the RyR blocker dantrolene ( Figure 9D) [67]. These findings together suggested a rescue of the arrhythmic phenotype by RyR2 block causing Nav1.5 rescue rather than Nav1.5 block. RyR2 inhibition would reduce the elevated diastolic Ca 2+ and its pro-arrhythmic inhibition of Nav1.5 [67]. The latter mechanism of action could underlie anti-arrhythmic effects of monotherapeutic low-dose flecainide introduced to treat clinical CPVT [62,[68][69][70][71].
Ca 2+ -mediated regulation of Nav1.5 may also contribute to commoner pro-arrhythmic cardiac conditions associated with spontaneous SR Ca 2+ leak. The latter was reported in peroxisome proliferator activated receptor-γ coactivator-1 (PGC-1) transcriptional coactivator deficient (Pgc1-β −/− ) murine models for pro-arrhythmic metabolic changes related to ageing, obesity and diabetes mellitus [72]. Atrial fibrillation, cardiac failure and hypertrophic cardiomyopathies are also accompanied by spontaneous SR Ca 2+ leak. Classically, SR Ca 2+ leak is implicated in a pro-arrhythmic activation of inward depolarizing, NCX current [44]. However, the pro-arrhythmic phenotypes in Pgc1-β −/− atria and ventricles were also associated with reduced I Na [73,74], (dV/dt) max and CVs [75,76]. A decreased I Na in these experimental conditions as well as in clinical heart failure or atrial fibrillation slowing action potential CV could contribute pro-arrhythmic substrate.

Perspectives
• Action potential generation by Na + channel (Nav) activation and the resulting release of intracellular Ca 2+ stores underly skeletal and cardiac myocyte excitation-contraction coupling abnormalities which underly a wide range of human genetic diseases.
• Nav channels possess sites directly or indirectly binding Ca 2+ potentially of regulatory importance in their reciprocal Ca 2+ -mediated feedback regulation. Evidence from cell expression systems, native myocytes and normal and disease models demonstrate such Ca 2+ -mediated Nav regulation effects.
• Future studies may correlate this molecular evidence bearing particularly on the Nav C-terminal and III-IV linker domains and biophysical studies of Na + channel function with associated clinical conditions.