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Review

Calmodulin Interactions with Voltage-Gated Sodium Channels

by
Xin Wu
and
Liang Hong
*
Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(18), 9798; https://doi.org/10.3390/ijms22189798
Submission received: 24 July 2021 / Revised: 6 September 2021 / Accepted: 7 September 2021 / Published: 10 September 2021
(This article belongs to the Special Issue Calmodulin Binding Proteins)
Browse Figures
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Abstract

:
Calmodulin (CaM) is a small protein that acts as a ubiquitous signal transducer and regulates neuronal plasticity, muscle contraction, and immune response. It interacts with ion channels and plays regulatory roles in cellular electrophysiology. CaM modulates the voltage-gated sodium channel gating process, alters sodium current density, and regulates sodium channel protein trafficking and expression. Many mutations in the CaM-binding IQ domain give rise to diseases including epilepsy, autism, and arrhythmias by interfering with CaM interaction with the channel. In the present review, we discuss CaM interactions with the voltage-gated sodium channel and modulators involved in CaM regulation, as well as summarize CaM-binding IQ domain mutations associated with human diseases in the voltage-gated sodium channel family.

Graphical Abstract

1. Introduction

The voltage-gated sodium channel (Nav) plays a vital role in the generation and propagation of action potential in excitable cells such as neurons and cardiac myocytes [1]. The family of voltage-gated sodium channels has nine members named Nav1.1 through Nav1.9 [2]. Among them, Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are predominantly expressed in neurons of the central nervous system [3,4]. Three isoforms (Nav1.7, Nav1.8, and Nav1.9) are widely expressed in neurons of the peripheral nervous system, such as dorsal root ganglia (DRG) neurons [5,6,7]. The isoform Nav1.4 is responsible for upstroke of the action potential in skeletal muscle [8], and Nav1.5 is a cardiac-specific isoform known as cardiac sodium channel (Figure 1) [9].
An essential property of voltage-gated sodium channels is “inactivation”, which prevents the reopening of the channel until complete recovery [10]. The inactivation regulates the frequency of action potential firing initiated by sodium channels in excitable cells. It reduces the breakdown of ionic gradients and cell death.
All voltage-gated sodium channels contain a calmodulin (CaM)-binding IQ domain necessary for the channel inactivation [11]. CaM is a small protein expressed in all eukaryotic cells [12]. It acts as a ubiquitous signal transducer and regulates essential processes such as neuronal plasticity, muscle contraction, and immune response [13]. CaM has two globular domains (i.e., N-terminal lobe and C-terminal lobe) connected by a linker. Each lobe contains two EF-hand motifs binding to Ca2+ [14]. In the Ca2+-free state, the EF-hands are collapsed in a compact configuration. When Ca2+ is bound to CaM, a conformational change occurs in the protein and rearranges the structural information of CaM.
The CaM-binding IQ domain is located within the C-terminal domain of the voltage-gated sodium channel. It contains around 25 residues with two highly conserved amino acids, isoleucine (I) and glutamine (Q), in the middle of the motif. The residues in the IQ domain form a seven-turn α-helix binding to CaM in a Ca2+-independent manner. In addition to the IQ domain, a globular domain in the C-terminus of voltage-gated sodium channel consists of EF hand-like (EFL) motifs that interact with CaM in the absence of Ca2+ [15,16], whereas an intracellular loop connecting domains III and IV (III-IV linker) in some isoforms of the sodium channel is shown to interact with CaM in the presence of Ca2+ [17].

2. CaM Regulation of Voltage-Gated Sodium Channel

CaM binds to the voltage-gated sodium channel and modulates the sodium channel gating process [15], alters sodium current density [18], and regulates sodium channel protein trafficking and expression [19].
One of the CaM modulations of voltage-gated sodium channel gating is inactivation (Figure 2). Voltage-gated sodium channel has two different types of inactivation, known as fast inactivation and slow inactivation. Fast inactivation occurs by occlusion of the intracellular pore by an “inactivation gate” formed by the cytoplasmic III-IV linker [10,20]. Slow inactivation is related to conformational rearrangements of the selectivity filter of the channel [21,22]. CaM mediates the fast inactivation by an interaction between III-IV linker and C-terminal domain of the voltage-gated sodium channel [23]. It enhances the slow inactivation by inducing a hyperpolarized shift of voltage dependence of inactivation and reducing the channel’s availability. CaM interaction with the sodium channel affects channel inactivation kinetics. It strengthens the development of inactivation and slows the recovery from inactivated state of the sodium channel.
In some isoforms of the voltage-gated sodium channels, CaM influences the activation process. It was reported that co-expression of CaM and Nav1.1 produced a hyperpolarized shift of the voltage dependence of activation of the Nav1.1 sodium channel [24].
Moreover, CaM can modulate sodium channel current density [18]. Studies reported that CaM regulation of sodium current density is Ca2+-dependent [24,25]. CaM does not affect sodium current amplitude in the absence of Ca2+. In contrast, it increases the current density in the presence of a high concentration of Ca2+.
In addition to modulation of the gating process of the voltage-gated sodium channel, CaM influences sodium channel protein trafficking and expression. It was shown to mediate the cell surface expression of Nav1.4 protein through the interaction with the IQ domain of the sodium channel [19].

3. Modulators Involved in CaM Regulation of Sodium Channel

There are several cellular partners involved in CaM regulation of the voltage-gated sodium channel function such as FHF (fibroblast growth factor homologous factor) [26]. CaMKII (calcium/calmodulin-dependent protein kinase II) [27], and cations (Ca2+, Mg2+) [11,28,29].
The most crucial regulator is Ca2+ [11,28]. In the presence of Ca2+, Ca2+/CaM (Ca2+-binding form of CaM) has a different regulation of sodium channel function compared with apo-CaM (Ca2+ free CaM), and Ca2+ binding to CaM induces rearrangements between the voltage-gated sodium channel and CaM lobes. In the absence of Ca2+, the N-lobe of CaM does not target the IQ domain (Figure 3a and Figure 4a,b). In the presence of Ca2+, the structure of the N-lobe is rearranged by Ca2+. It generates an allosterically conformational change binding to the distal IQ domain of the channel (Figure 3b and Figure 4c). However, the C-lobe of CaM can bind to the voltage-gated sodium channel IQ domain in a Ca2+-independent manner.
The FHFs were shown to form a complex with CaM and modulate CaM regulation of Nav1.5 function [26]. A recent study discovered fibroblast growth factor homologous factor FGF13 (fibroblast growth factor 13, also known as FHF2) tuned arrhythmogenic late sodium currents in CaM binding-deficient channels in cardiac myocytes [26]. The crystal structures in Figure 4 showed that FGF and Ca2+ mediated the interaction between CaM and Nav1.5 IQ domain. It indicated that the N-lobe of CaM interacted with the EFL of the channel in the absence of FGF. However, when FGF was bound to the channel, the N-lobe of CaM was rearranged. It did not make contact with the EFL and was free to interact with other parts of the channel complex. The N-lobe of CaM likely interacted with the III-IV linker of the sodium channel and modulated the channel’s inactivation.
In addition, CaMKII has been reported to influence CaM interaction with the sodium channel. One study found CaMKII-mediated phosphorylation of the Nav1.1 sodium channel IQ domain increased CaM binding affinity to the channel [27]. Three phosphorylation sites (T1909, S1918, and T1934) were identified in the Nav1.1 IQ domain. Other studies demonstrated that CaMKII phosphorylated Nav1.5 at residue S571 to decrease channel availability. The CaMKII phosphorylation site S571 is located at the intracellular DI-DII loop of the Nav1.5 sodium channel, and mutation at position S571 abolished the hyperpolarized shift of the voltage dependence of inactivation produced by CaMKII phosphorylation. The effects of CaMKII-mediated phosphorylation of IQ domain on sodium currents have not been characterized. CaMKII-dependent IQ domain phosphorylation might confer normal sodium currents important for the function of cells where the Nav channels are expressed. The CaMKII-mediated effect seems to be isoform-specific. CaMKII phosphorylation enhances CaM affinity for sodium channel Nav1.1 but not for Nav1.2 [27,30].
Moreover, Mg2+ is involved in the CaM-mediated modulation. In some neurons, low Mg2+ treatment increases the effects of CaM on the voltage-gated sodium channel activity [29].
With these partners, CaM regulates voltage-gated sodium channel function by altering channel gating, sodium current density, and expression of the sodium channel protein. Studies have shown that CaM has isoform-specific modulation of voltage-gated sodium channel function [31,32,33,34].

4. Interaction between CaM and Sodium Channel Isoforms

4.1. Nav1.1

Nav1.1 is one of the voltage-gated sodium channels predominantly expressed in the central nervous system [35]. It is known to be highly expressed in the soma and apical dendrite of the Purkinje cells.
The effect of CaM on Nav1.1 channel function is Ca2+-dependent. CaM does not affect sodium current density of wild-type Nav1.1 channel in the absence of Ca2+ [25]. but significantly increases Nav1.1 current density in the presence of 10µM intracellular Ca2+ [24].
CaM mediates Nav1.1 channel gating process when binding to the IQ domain, and overexpression of CaM induces a hyperpolarized shift of the voltage dependence of activation of Nav1.1 [24]. However, it does not generate significant effects on the voltage dependence of inactivation at high intracellular Ca2+ concentration but accelerates inactivation process of Nav1.1 with low Ca2+ [24].
A GST pull-down assay reported CaM interaction with Nav1.1 sodium channel IQ domain [27]. The Nav1.1 IQ domain preferentially binds to apoCaM than Ca2+/CaM. In the absence of Ca2+, C-lobe of CaM is the predominant domain binding to Nav1.1, and N-lobe of CaM is proposed to interact with other parts of the channel [36]. In the presence of Ca2+, N-lobe of CaM is shown to be the predominant domain binding to Nav1.1 [27]. Moreover, one study reported that CaMKII-mediated phosphorylation of Nav1.1 enhanced CaM interaction with IQ domain of the Nav1.1 channel [27].

4.2. Nav1.2

The neuronal sodium channel Nav1.2 is expressed in granule cells and interneurons in the central nervous system [37], and plays roles in excitatory neurons in the neocortex and hippocampus [38].
CaM has been shown to mediate Ca2+-dependent regulation of the Nav1.2 channel [39]. CaM binding to Nav1.2 reduces Ca2+ binding affinity in the CaM-binding sites [40]. Although there is a Ca2+-binding EFL motif upstream of the IQ domain in the Nav1.2 channel, the Ca2+-mediated changes in modification of Nav1.2 function are proposed to be likely from the interaction between CaM and Nav1.2 IQ domain when Ca2+ binding to CaM. The Nav1.2 channel EFL motif was indicated to enhance Ca2+ binding to CaM interacting with the channel [41].
Structural study analysis showed the C-lobe of CaM anchored to the Nav1.2 channel IQ domain independent of Ca2+ concentration. In the absence of Ca2+, the N-lobe of CaM did not interact with the IQ domain. When binding to Ca2+, the N-lobe of CaM was induced to interact with the distal IQ domain of the Nav1.2 channel (Figure 3) [42].
CaM has no notable effects on the Nav1.2 current density. Overexpression of CaM does not significantly decrease the peak sodium current amplitude of the Nav1.2 channel [43].

4.3. Nav1.3

Although Nav1.3 is a neuronal sodium channel [44], the first paper reporting CaM regulation of Nav1.3 is from a study on microvessels of the kidney [45].
The study examined the expression of Nav1.3 in rat descending vasa recta, a series of blood vessels that perfuse the renal medulla. Further, it verified that CaM binding to the C-terminus of Nav1.3 by pull-down and immunoprecipitation assays [45]. The voltage-gated sodium currents in the vasa recta pericytes were remarkably suppressed by CaM inhibitors CIP (calmodulin inhibitory peptide) and W7 (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride). However, CIP or W7 did not generate alteration of gating process of endogenous voltage-gated sodium currents in vasa recta cells [45].
Another study in hippocampal neurons showed that CaM regulates Nav channel function in an Mg2+-dependent manner. The Nav1.3 activities, together with Nav1.1 and Nav1.2, were more sensitive to Ca2+/CaM regulation in a low-Mg2+ environment than normal neurons [29].

4.4. Nav1.4

Nav1.4 is the predominant subtype of sodium channel initiating skeletal muscle action potential [8]. Nav1.4 mutations associated with periodic paralysis disorders affect skeletal muscle excitability [46].
CaM influences the gating process of the Nav1.4 sodium channel, and the effects of CaM on Nav1.4 inactivation are related to Ca2+ [31]. In the presence of Ca2+, CaM shifts the voltage dependence of inactivation of the channel to hyperpolarizing direction. When the free Ca2+ is removed, the CaM-induced shift of the steady-state inactivation curve is attenuated [31].
CaM regulates Nav1.4 sodium current density. The decreased sodium currents caused by Nav1.4 IQ mutations were shown to be rescued by overexpression of CaM. However, CaM does not significantly affect the sodium current density of the wild-type Nav1.4 channel [32].
In addition to functional regulation, CaM influences Nav1.4 channel trafficking and expression. One study demonstrated an intimate relationship between intact Nav1.4 channels and CaM in live cells and revealed that CaM participated in the regulation of cell surface expression of Nav1.4 protein through the interaction with the Nav1.4 channel IQ domain [19].

4.5. Nav1.5

The most well-studied voltage-gated sodium channel interacting with CaM is the Nav1.5. The cardiac sodium channel Nav1.5, encoded by the SCN5A gene, plays a critical role in the fast depolarization of the cardiac action potential [47]. Cardiac sodium channel dysfunction caused by Nav1.5 mutations was reported to remodel abnormal action potential underlying arrhythmias [48].
CaM binds to Nav1.5 C-terminal domain and enhances the inactivation of Nav1.5 channel. It shifts the steady-state inactivation to hyperpolarization and influences inactivation kinetic [49]. CaM mediates Ca2+ regulation of Nav1.5 sodium channel function [50]. In the presence of Ca2+, CaM is promoted to bind to the Nav1.5 inactivation gate. Perturbation of the interaction between the gate and CaM generates a decreased recovery from inactivation of the Nav1.5 channel [51,52].
The sodium channel Nav1.5 IQ domain mutations associated with arrhythmia reduce CaM binding affinity. In HEK293 cells, overexpression of CaM attenuates late sodium currents caused by Nav1.5 IQ domain mutations [43]. CaM binding to Nav1.5 channel IQ domain also modifies late sodium currents in cardiac myocytes [26]. Study in transgenic mouse models showed FHFs tuned cardiac late sodium currents in ventricular myocytes. FHFs diminished late sodium current of the Nav1.5 channel IQ/AA mutation (substitution of two conserved residues isoleucine (I) and glutamine (Q) with double alanines (AA) in the IQ domain), IQ/AA mutation has been reported to disrupt CaM binding to the IQ domain of voltage-gated sodium channels.
Studies in guinea-pig ventricular myocytes revealed CaM enhances the cardiac sodium current density [53]; this is different from the result that CaM does not influence on sodium current amplitude when co-expressing with Nav1.5 channel in HEK293 cells [43]. It indicates that some environmental factors in cardiac tissues are involved in CaM regulation of Nav function.
Additionally, a recent study proposed CaM can bind to the N-terminal domain of the cardiac Nav1.5 channel, wherein one Brugada syndrome-associated mutation in the N-terminal domain of Nav1.5 was shown to weaken the interaction between CaM and Nav1.5 N-terminal domain [54].

4.6. Nav1.6

Nav1.6 is the primary voltage-gated sodium channel at the myelin-sheath gaps. It is important in the generation and propagation of the action potential along myelinated axons [55].
CaM binding to Nav1.6 is crucial for functional sodium current expression [32]. Disruption of CaM binding to the Nav1.6 channel significantly reduces sodium current amplitude. Overexpression of CaM rescues the decreased sodium currents caused by Nav1.6 channel CaM-binding domain mutations. CaM enhances the rate of Nav1.6 inactivation and switches Nav1.6 channel from fast mode to slow mode. It has no effects on the voltage dependence of activation of Nav1.6 [32].
The structural study reported that CaM interacted with different residues of the Nav1.6 channel, depending on the absence or presence of Ca2+. However, three key residues (Arg1902, Tyr1904, and Arg1905) in the Nav1.6 channel IQ domain were identified to interact with CaM in a Ca2+-independent manner [56].

4.7. Nav1.7

The voltage-gated sodium channel Nav1.7 is highly expressed in nociceptive dorsal root ganglion (DRG) neurons and superior cervical ganglion (SCG) neurons [57].
The electrophysiological study on CaM interacting with Nav1.7 has not yet been determined. One nuclear magnetic resonance (NMR) study analyzed complexes of calcium-free CaM bound to peptides of IQ motifs of Nav1.7. It showed C-lobe of CaM contributed to the interface with the IQ motif of Nav1.7 and the N-lobe of CaM interacted with other parts of the sodium channel [36]. These results are consistent with findings in other voltage-gated sodium channels and suggest that an interaction interface between CaM and IQ domain of voltage-gated sodium channels is highly conserved in the C-lobe of CaM.

4.8. Nav1.8

Nav1.8 channel is expressed in dorsal root ganglia (DRG) neurons [6], and genetic research has shown this channel is also implicated in cardiac function [58], in which Nav1.8 was identified in cardiomyocytes and intracardiac neurons, contributing to cardiac repolarization [59].
CaM is a functional partner of Nav1.8 sodium and interacts with Nav1.8 in vivo [18]. It was found that CaM can coimmunoprecipitate with endogenous Nav1.8 channels from native DRG. CaM mediated a frequency-dependent inhibition of sodium channels in the DRG neurons. The sodium currents in neurons were significantly decreased in the presence of high cellular Ca2+ (10 µM free Ca2+) and CaM antagonist calmodulin-binding peptide (CBP), suggesting CaM modulated Nav1.8 current density in neurons [18].
A recent study demonstrated the CaM enhanced slow inactivation of the Nav1.8 channel and reduced channel availability. It modulated Nav1.8 function through its interaction with the IQ domain. CaM mediated regulation was abrogated in mutations disrupting CaM binding to the channel [60].

4.9. Nav1.9

Nav1.9 is expressed in sensory neurons of the DRG and trigeminal ganglion [61].
To date, there is no study investigating the interaction between CaM and Nav1.9 channel. The Nav1.9 IQ domain shares high sequence homology with other sodium channels (Figure 5). It remains to be determined if CaM regulation of Nav1.9 channel plays a role in channel function in sensory neurons.

5. CaM-Binding IQ Domain Mutations in Voltage-Gated Sodium Channel

A large number of voltage-gated sodium channel mutations have been linked with disorders of the nervous and cardiovascular systems [62]. Some mutations are located in the highly conserved CaM-binding IQ domain and cause severe neurological diseases and cardiac arrhythmia (Figure 5, Table 1).

5.1. Nav1.1 Mutations

Nav1.1 mutations are associated with familial epilepsies. The Nav1.1-I1922T was identified in patients with Dravet syndrome [63,64]. In this mutation, highly conserved residue isoleucine (I) that plays a critical role in CaM binding to the IQ domain was substituted by threonine (T). Another mutation, Nav1.1-R1928G, is linked with cryptogenic epileptic syndrome [65], and characterized in patients diagnosed with generalized epilepsy with febrile seizures plus (GEFS+) [66], familial hemiplegic migraine (FHM) [67], and severe myoclonic epilepsy of infancy (SMEI) [68].
Additionally, CaM was reported to generate partially functional rescue of Nav1.1-M1841T, an epileptogenic mutation located upstream of the CaM-binding IQ domain of the Nav1.1 channel [25]. M1841T was characterized as a loss-of-function mutant and generated very small sodium currents. Co-expression of CaM with M1841T partially rescued the loss of function of the channel and induced a 3.5-fold increase in sodium current amplitude [25].

5.2. Nav1.2 Mutations

The Nav1.2 mutation R1902C has been identified in patients with familial autism [69]. Nav1.2-R1902C perturbed Ca2+-dependent changes in the regulation of Nav1.2 function [39]. Protein pull-down assays showed R1902C abolished Ca2+-dependence of CaM binding [30]. In the presence of Ca2+, Nav1.2-R1902C induced a hyperpolarization shift of the voltage dependence of activation and inactivation of the channel [42].
The mutation Nav1.2-R1918H was associated with idiopathic generalized epilepsy [70]. This mutation produced a pathogenically increased late sodium current, indicating a gain-of-function mechanism in Nav1.2 mutations associated with epilepsy. The enhanced late sodium current mediated by Nav1.2-R1918H was able to be rescued by CaM overexpression [43].

5.3. Nav1.5 Mutations

The Nav1.5-R1898H is a mutation located at the N-terminal end of the CaM-binding IQ domain of the Nav1.5 channel and was characterized in a patient with arrhythmogenic right ventricular dysplasia/cardiomyopathy (AVRD/C) [71]. A study assessed this mutation’s cellular and molecular phenotype using an induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) approach. It showed that R1898H was a loss-of-function mutation that caused around 40% reduction in peak sodium current [71]. Three-dimensional super-resolution fluorescence microscopy (3D-SRFM) experiments showed a structural deficit of Nav1.5 and N-cadherin clusters in the R1898H mutation cells, indicating a role that Nav1.5 dysfunction associated with R1898H mediated cardiomyopathy [71]. Another mutation at the same position, R1898 (Nav1.5-R1898C), was identified in a patient with Brugada syndrome [72]. It should be noted that the Nav1.5-R1898C corresponds to the autism mutation Nav1.2-R1902C (Figure 5). In Nav1.2, the mutation (Nav1.2-R1902C) affected the gating process of the channel and perturbed Ca2+-dependent regulation of Nav1.2 function [42]. It is likely that the same mutation in the Nav1.5 (Nav1.5-R1898C) generated similar consequences on the Nav1.5 function and played similar roles in cardiac diseases.
There are two mutations at the position Nav1.5-E1901 [73,74]. The Nav1.5-E1901K was associated with Brugada syndrome [74], and Nav1.5-E1901Q was identified in a patient with type 3 of the long-QT syndrome (LQT3). Nav1.5-E1901Q was shown to cause an increased late sodium current [73]. In the absence of Ca2+, the negative residue Glu at the position 1901 (E1901) might couple with a positive residue of CaM to form charge–charge interaction, stabilizing CaM binding to the IQ domain of the channel [75]. Replacement of Glu with Gln (E1901Q) or Lys (E1901K) likely disrupted the salt bridge formed by the charge–charge interaction between CaM and Nav1.5 IQ domain and produced a perturbation of CaM regulation of the channel, providing an explanation for the pathological roles of those mutations in Brugada syndrome and LQT3 syndrome.
The Nav1.5 mutation S1904L was associated with Brugada and long-QT syndromes [74,76]. The position of Ser1904 was proposed to use hydrogen bonds to interact with the main chain of the C-lobe of the CaM. Mutation at the position of 1904 (S1904L) perturbed the Nav1.5 channel interaction with CaM. In the absence of Ca2+, a flow cytometric FRET two-hybrid analysis between the S1904L and CaM demonstrated a weaker affinity compared to the wild-type Nav1.5 channel [15]. Additionally, S1904L induced an enhanced late sodium current, indicating a delayed inactivation of the channel caused by CaM dysregulation of the channel in this mutation [76].
Nav1.5-Q1909R was linked with sudden infant death syndrome (SIDS) and LQT3 syndrome [77,78]. Functional studies showed that Q1909R is a gain-of-function mutation [79]. Ventricular action potential (AP) simulations showed a frequency-dependent reduction of AP duration in Q1909R carriers [79]. Moreover, Q1909R resulted in an increase in late sodium current. Co-expression of CaM or higher intracellular Ca2+ reduced the enhanced late sodium currents [43,79].
The mutation Nav1.5-R1913H was identified in a patient with LQT3 syndrome [80]. In the absence of Ca2+, the position Arg1913 interacts with negative residues of C-lobe of CaM. Mutation at this position (R1913H) resulted in an enhanced late sodium current, and overexpression of CaM can reduce the enhanced late sodium current [43]. Another mutation (R1919C) was reported to be associated with Brugada syndrome and Long-QT syndrome [81].
A Brugada syndrome mutation A1924T is located at the C-terminal end of the IQ domain. Nav1.5-A1924T induced a negative shift in voltage-state activation, which would cause some persistent depolarization of the channel [82]. Nav1.5-A1924T showed a reduced slow inactivation, and application of CaM rescued the decreased slow inactivation caused by Nav1.5-A1924T [49].
In addition to IQ domain mutations in the cardiac Nav1.5 channel, there are several mutations in other domains of Nav1.5 involved in the regulation of CaM interaction with the sodium channel. In the N-terminal domain of the Nav1.5 channel, one Brugada syndrome mutation R121W was reported to weaken the interaction between CaM and the channel [54]. In the III-IV linker of the Nav1.5 channel, Brugada syndrome mutation K1493del (a Lys was deleted at the position 1493) decreased Ca2+-dependent CaM-interaction with Nav1.5 channel [83]. Brugada syndrome mutations Y1494N and I1521K were shown to cause domain-specific perturbations of the interaction with CaM [51]. In the EF hand-like (EFL) motifs upstream of the IQ domain of Nav1.5 channel, mutations (L1825P and Y1795insD) associated with LQT and Brugada syndromes perturbed CaM regulation of the channel function [84]. The persistent sodium currents produced by another Brugada syndrome mutation E1784K in the EFL motif were modulated by CaM overexpression [23]. Moreover, the mutation F1759A near the EFL motifs tuned the late sodium currents in CaM binding-deficient channels in cardiac myocytes [26].

5.4. Nav1.8 Mutations

Nav1.8 channel IQ domain mutations were reported to be linked with cardiac arrhythmias. One clinical research reported Nav1.8-R1863Q mutation in a patient with Brugada syndrome (BrS) [85]. The Nav1.8 mutation R1869C was identified in an index case with atrial fibrillation (AF) and BrS [86]. In addition, mutation Nav1.8-R1869G was identified to co-segregate with familial AF [87].
All IQ domain mutations influence CaM interaction with the Nav1.8 channel. Nav1.8-R1863Q reduced the CaM-induced hyperpolarization shift of the voltage dependence of inactivation of the channel. Nav1.8-R1869C and Nav1.8-R1869G disrupted CaM-induced hyperpolarization shift and attenuated effects of CaM on development and recovery from slow inactivation [60]. These results suggested that Nav1.8 IQ domain mutations weakened the interaction between CaM and Nav1.8 channel and perturbed CaM regulation of Nav1.8 function.

6. Conclusions and Perspectives

We have described the interaction between CaM and each isoform of voltage-gated sodium channel family and summarized mutations associated with human diseases in CaM regulation of voltage-gated sodium channels.
The effects of CaM on the sodium channels are complex. They are not only involved in various regulation ways but also related to multiple factors in the cells. CaM regulates Nav channel gating by binding to the IQ motif of the channel. Intracellular Ca2+ is also reported to modulate cardiac Nav1.5 channel inactivation by binding to an EF hand-like (EFL) motif of the sodium channel. The multiple binding sites of Ca2+ (either in the EFL of Nav channel or in the EF-hand of CaM) and ability of CaM binding to the IQ domain of the Nav channel result in various regulations of Ca2+ and/or CaM on the Nav channel function. Therefore, delineating Nav modulation by CaM will require developing novel approaches that exclude or minimize other factors’ modulation on CaM regulation of sodium channel function. In addition, studies have shown that CaM has isoform-specific modulation of voltage-gated sodium channels [31,32,33,34]. Because sodium channel isoforms have specific tissue distribution (Figure 1), whether environmental factors in different tissues are involved in CaM regulation of Nav function remains unclear. Studies in the field will help explore the regulatory mechanism underlying distinct properties of CaM modulation of voltage-gated sodium channel function. Moreover, although the canonical binding site of CaM is the C-terminal domain of the sodium channel, a recent study showed another interaction site between CaM and the channel. A putative CaM-binding sequence comprising 26 amino acids is located at the N-terminal domain of the sodium channel [54]. Future studies on the complexes of a Nav N-terminal domain and CaM will extend and provide a complete understanding of the contribution of CaM on the Nav function.
In summary, CaM plays a critical regulatory role in cellular electrophysiology by its ability to bind to voltage-gated sodium channels. Understanding how it regulates sodium channel function is a crucial step towards developing treatments for diseases associated with sodium channel mutations.

Funding

This work was supported in part by the National Institute of Health Grant R01GM139991 (L.H.) and the American Heart Association Grant 19CDA34630041 (L.H.).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

NavVoltage-gated sodium channel
CaMCalmodulin
Apo-CaMCa2+-free CaM
Ca2+/CaMCa2+-binding CaM
IQ domainA domain with highly conserved residues isoleucine (I) and glutamine (Q)
CBPCalmodulin-binding peptide
CIPCalmodulin inhibitory peptide
EFLEF hand-like motifs
FHFFibroblast growth factor homologous factor
FGFFibroblast growth factor
CaMKIICalcium/calmodulin-dependent protein kinase II
APAction potential
DRGDorsal root ganglia
SCGSuperior cervical ganglion
NMRNuclear magnetic resonance
GEFS+Generalized epilepsy with febrile seizures plus
FHMFamilial hemiplegic migraine
SMEISevere myoclonic epilepsy of infancy
SIDSSudden infant death syndrome
LQT3Type 3 of long-QT syndrome

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Figure 1. A phylogenetic tree and tissue distribution of voltage-gated sodium channels. Isoforms predominantly expressed in the central nervous system (blue), peripheral nervous system (green), skeletal muscle (red), and cardiac muscle (orange) are highlighted.
Figure 1. A phylogenetic tree and tissue distribution of voltage-gated sodium channels. Isoforms predominantly expressed in the central nervous system (blue), peripheral nervous system (green), skeletal muscle (red), and cardiac muscle (orange) are highlighted.
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Figure 2. CaM regulation of sodium channel function by interaction with the IQ domain. (a-c) Voltage-gated sodium channel (Nav) has three states known as “close” (a), “open” (b), and “inactivation” (c). Several functional parts (inactivation gate, activation gate, and selectivity filter) are involved in channel gating. The “+”and ”-” represent charge separation across the membrane. Differences in charges on opposite sides of the cellular membrane generate the membrane potential. At rest, the activation gate is closed (a). When the membrane is depolarized, the activation gate opens and allows Na+ to enter (b). At the same time, depolarization of the membrane causes the inactivation gate to close and induces the fast inactivation. The channel also shows slow inactivation related to conformational rearrangements of the selectivity filter (highlighted in yellow arrows in c). (d–f) CaM binds to the voltage-gated sodium channel IQ domain and modulates the sodium channel inactivation. CaM affects channel’s fast inactivation kinetics (e) by an interaction between the inactivation gate formed by cytoplasmic III-IV linker and the C-terminal domain of the channel (d). CaM enhances the slow inactivation (highlighted in bigger yellow arrows in d). It induces a hyperpolarized shift of voltage dependence of inactivation and reduces the channel’s availability (f).
Figure 2. CaM regulation of sodium channel function by interaction with the IQ domain. (a-c) Voltage-gated sodium channel (Nav) has three states known as “close” (a), “open” (b), and “inactivation” (c). Several functional parts (inactivation gate, activation gate, and selectivity filter) are involved in channel gating. The “+”and ”-” represent charge separation across the membrane. Differences in charges on opposite sides of the cellular membrane generate the membrane potential. At rest, the activation gate is closed (a). When the membrane is depolarized, the activation gate opens and allows Na+ to enter (b). At the same time, depolarization of the membrane causes the inactivation gate to close and induces the fast inactivation. The channel also shows slow inactivation related to conformational rearrangements of the selectivity filter (highlighted in yellow arrows in c). (d–f) CaM binds to the voltage-gated sodium channel IQ domain and modulates the sodium channel inactivation. CaM affects channel’s fast inactivation kinetics (e) by an interaction between the inactivation gate formed by cytoplasmic III-IV linker and the C-terminal domain of the channel (d). CaM enhances the slow inactivation (highlighted in bigger yellow arrows in d). It induces a hyperpolarized shift of voltage dependence of inactivation and reduces the channel’s availability (f).
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Figure 3. Ca2+ modulates the interaction between CaM and Nav1.2 IQ domain. The NMR structures showed Nav1.2 IQ domain (blue) in complex with CaM (green) in the absence (a) or presence (b) of Ca2+. Two lobes (N-lobe and C-lobe) of CaM are indicated. C-lobe of CaM bound to the Nav1.2 channel IQ domain independent of Ca2+ concentration. In the absence of Ca2+, the N-lobe of CaM did not interact with the IQ domain (PDB: 2KXW) (a). When binding to Ca2+, the N-lobe of CaM was induced to interact with the distal IQ domain of Nav1.2 channel (PDB: 2M5E) (b).
Figure 3. Ca2+ modulates the interaction between CaM and Nav1.2 IQ domain. The NMR structures showed Nav1.2 IQ domain (blue) in complex with CaM (green) in the absence (a) or presence (b) of Ca2+. Two lobes (N-lobe and C-lobe) of CaM are indicated. C-lobe of CaM bound to the Nav1.2 channel IQ domain independent of Ca2+ concentration. In the absence of Ca2+, the N-lobe of CaM did not interact with the IQ domain (PDB: 2KXW) (a). When binding to Ca2+, the N-lobe of CaM was induced to interact with the distal IQ domain of Nav1.2 channel (PDB: 2M5E) (b).
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Figure 4. FGF and Ca2+ modulate the interaction between CaM and Nav1.5 IQ domain. (a) The structure of the complex of the C-terminal domain of Nav1.5 with CaM showed C-lobe of CaM bound to the IQ domain, and N-lobe of CaM interacted with EFL (PDB: 4OVN). (b) Crystal structure showed the interactions between the C-terminal domain of Nav1.5, FGF, and CaM (PDB: 4DCK). When FGF bound to the channel, the N-lobe of CaM was rearranged. It did not make contact with EFL and was free to interact with other parts of the channel complex. The N-lobe of CaM likely interacted with the inactivation gate of the sodium channel and modulated the channel’s inactivation. (c) In a high concentration of Ca2+, the N-lobe of CaM was induced to interact with the distal IQ domain of Nav1.5 channel (PDB: 4JQ0).
Figure 4. FGF and Ca2+ modulate the interaction between CaM and Nav1.5 IQ domain. (a) The structure of the complex of the C-terminal domain of Nav1.5 with CaM showed C-lobe of CaM bound to the IQ domain, and N-lobe of CaM interacted with EFL (PDB: 4OVN). (b) Crystal structure showed the interactions between the C-terminal domain of Nav1.5, FGF, and CaM (PDB: 4DCK). When FGF bound to the channel, the N-lobe of CaM was rearranged. It did not make contact with EFL and was free to interact with other parts of the channel complex. The N-lobe of CaM likely interacted with the inactivation gate of the sodium channel and modulated the channel’s inactivation. (c) In a high concentration of Ca2+, the N-lobe of CaM was induced to interact with the distal IQ domain of Nav1.5 channel (PDB: 4JQ0).
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Figure 5. Alignment of voltage-gated sodium channel family IQ domain and CaM-binding IQ domain mutations. Sodium channel mutations in the IQ domain associated with human diseases are highlighted in yellow. The conserved residues Ile (I) and Glu (Q) are indicated by stars.
Figure 5. Alignment of voltage-gated sodium channel family IQ domain and CaM-binding IQ domain mutations. Sodium channel mutations in the IQ domain associated with human diseases are highlighted in yellow. The conserved residues Ile (I) and Glu (Q) are indicated by stars.
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Table
Table 1. CaM-binding IQ domain mutations in Nav channels.
Table 1. CaM-binding IQ domain mutations in Nav channels.
Pathological Mutation in CaM-Binding IQ DomainChange in NucleotidePhenotypeEffect On Nav Function
Nav1.1-I1922Tc.5765T>CDravet syndromeunknown
Nav1.1-R1928Gc.5782C>GFamilial epilepsy syndrome
Familial hemiplegic migraine
Cryptogenic epileptic syndrome
Dravet syndrome		unknown
Nav1.2-R1902Cc.5704C>TFamilial autism(1) Abolished Ca2+-dependence of CaM binding; (2) Induced hyperpolarization shift of the voltage dependence of activation and inactivation of the channel.
Nav1.2-R1918Hc.5753G>AIdiopathic generalized epilepsy (1) Induced an increased late sodium current; (2) Overexpression of CaM reduced the late sodium current.
Nav1.5-R1898Cc.5692C>TBrugada syndromeunknown
Nav1.5-R1898Hc.5693G>AArrhythmogenic right ventricular dysplasia/cardiomyopathy(1) Caused a reduction in peak sodium current; (2) Caused a structural deficit and decreased abundance of Nav1.5 and N-Cadherin clusters.
Nav1.5-E1901Kc.5701G>ABrugada syndromeunknown
Nav1.5-E1901Qc.5701G>CLong QT syndrome(1) Induced an increased late sodium current; (2) Overexpression of CaM reduced the late sodium current.
Nav1.5-S1904Lc.5711C>TLong QT syndrome
Brugada syndrome		(1) Reduced binding affinity between channel and CaM; (2) Induced an increased late sodium current.
Nav1.5-Q1909Rc.5726A>G Long QT syndrome
Sudden infant death syndrome		(1) Induced an increased late sodium current; (2) Overexpression of CaM reduced the late sodium current.
Nav1.5-R1913Hc.5738G>ALong QT syndrome(1) Induced an increased late sodium current; (2) Overexpression of CaM reduced the late sodium current.
Nav1.5-R1919Cc.5755C>TLong QT syndrome
Brugada syndrome		unknown
Nav1.5-A1924Tc.5770G>ABrugada syndrome(1) Induced a hyperpolarization shift in voltage-state activation; (2) Generated a reduced slow inactivation that was rescued by CaM.
Nav1.8-R1863Qc.5588 G>ABrugada syndrome(1) Reduced CaM-induced hyperpolarization shift of the voltage dependence of inactivation of the channel.
Nav1.8-R1869Cc.5605 C>TBrugada syndrome (1) Disrupted CaM-induced hyperpolarization shift; (2) Attenuated effects of CaM on development and recovery from slow inactivation.
Nav1.8-R1869Gc.5605 C>GAtrial fibrillation(1) Disrupted CaM-induced hyperpolarization shift; (2) Attenuated effects of CaM on development and recovery from slow inactivation.
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Wu, X.; Hong, L. Calmodulin Interactions with Voltage-Gated Sodium Channels. Int. J. Mol. Sci. 2021, 22, 9798. https://doi.org/10.3390/ijms22189798

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Wu X, Hong L. Calmodulin Interactions with Voltage-Gated Sodium Channels. International Journal of Molecular Sciences. 2021; 22(18):9798. https://doi.org/10.3390/ijms22189798

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Wu, Xin, and Liang Hong. 2021. "Calmodulin Interactions with Voltage-Gated Sodium Channels" International Journal of Molecular Sciences 22, no. 18: 9798. https://doi.org/10.3390/ijms22189798

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Wu, X., & Hong, L. (2021). Calmodulin Interactions with Voltage-Gated Sodium Channels. International Journal of Molecular Sciences, 22(18), 9798. https://doi.org/10.3390/ijms22189798

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