Sodium channelopathies of skeletal muscle and brain

Abstract

Voltage-gated sodium channels initiate action potentials in nerve, skeletal muscle, and other electrically excitable cells. Mutations in them cause a wide range of diseases. These channelopathy mutations affect every aspect of sodium channel function, including voltage sensing, voltage-dependent activation, ion conductance, fast and slow inactivation, and both biosynthesis and assembly. Mutations that cause different forms of periodic paralysis in skeletal muscle were discovered first and have provided a template for understanding structure, function, and pathophysiology at the molecular level. More recent work has revealed multiple sodium channelopathies in the brain. Here we review the well-characterized genetics and pathophysiology of the periodic paralyses of skeletal muscle and then use this information as a foundation for advancing our understanding of mutations in the structurally homologous α-subunits of brain sodium channels that cause epilepsy, migraine, autism, and related comorbidities. We include studies based on molecular and structural biology, cell biology and physiology, pharmacology, and mouse genetics. Our review reveals unexpected connections among these different types of sodium channelopathies.

Keywords: autism; epilepsy; migraine; periodic paralysis; sodium channels.

Graphical abstract

FIGURE 1.

Structure of voltage-gated sodium channels revealed step by step over >30 yr. A: cartoon model of purified brain sodium channels with α- and β-subunits circa 1986. ScTx, scorpion toxin; TTX, tetrodotoxin; P, protein phosphorylation. B: transmembrane folding diagram of the sodium channel α-subunit with key functional domains indicated circa 2000. Blue circle with h, inactivation particle with isoleucine, phenylalanine, methionine (IFM) motif; empty blue circles, inactivation gate receptor. C: structure of the inactivation gate peptide of the brain Na_V1.2 channel determined by NMR. D: top view of the structure of Na_VAb determined by X-ray crystallography circa 2011. Blue, pore module; green, voltage sensors. E: structure of the pore of Na_VAb. F: structure of the voltage sensor of Na_VAb. G: structure of Na_V1.4 with β₁-subunit circa 2018. The domain III–IV linker (brown) serves as the fast inactivation gate. H: close-up of the IFM motif of the inactivation gate (brown) interacting with its receptor. A: adapted from Ref. with permission from Annual Review of Biochemistry. B and C: adapted from Ref. with permission from Neuron. D–F: adapted from Ref. with permission from Nature. G and H: adapted from Ref. with permission from Science.

FIGURE 2.

Functional properties of sodium channels. Functional properties and their modifications induced by mutations involved in channelopathies are studied by performing voltage-clamp experiments that follow the general methods introduced by Hodgkin and Huxley in their seminal work (39). In modern experiments aimed at identifying functional effects of mutations, sodium channels are often expressed in transfected cell lines that do not have endogenous channels of interest, and currents are evoked controlling the membrane potential in voltage-clamp mode by means of the whole cell configuration of the patch-clamp technique. A: representative recordings of families of Na_V1.1 sodium currents with the 2-pulse voltage protocol shown at top. Inward sodium currents are a negative quantity by convention and therefore plotted downward. The first pulse is a step depolarization at different potentials (up to +20 mV) activating inward sodium currents (inset a), which then undergo fast inactivation during the 100-ms depolarization. These recordings determine the maximal current amplitude and the kinetics of fast inactivation from the open state within a few milliseconds. Current amplitude can be increased by gain-of-function mutations (depicted by green vertical arrow) or decreased by loss-of-function mutations (red vertical arrow). Sodium currents can be prolonged by gain-of-function mutations (green horizontal arrow) or shortened by loss-of-function mutations (red horizontal arrow). The small fraction of current with slower inactivation is called persistent sodium current (inset b, in which a single trace is shown). Gain-of-function mutations can increase its amplitude, whereas loss-of-function mutations can decrease it. The second pulse at 0 mV evokes sodium currents (inset c) whose amplitude decreases according to the amount of fast inactivation induced by the first pulse. Scale bars refer to the black traces. Similar voltage protocols with longer depolarizations (up to tens of seconds) can be used to study slow inactivation. B: the activation curve (orange) is obtained by plotting the normalized peak conductance (G/G_max) of the currents in inset a as a function of the stimulus potential. The fast inactivation curve (violet) is obtained by plotting the normalized peak values of the currents (I/I_max) displayed in inset c as a function of the potential of the first pulse. Gain- and loss-of-function mutations can induce shifts of the curves (horizontal arrows). The current elicited at membrane potentials in which activation and inactivation curves overlap (yellow area) is called window current. When there is persistent current, the inactivation curve shows a baseline at positive voltages (solid line, 0% persistent current; dashed line, 5% persistent current). C: the persistent current can also be elicited with slow depolarizing voltage ramps that inactivate the fast transient current; of note, relatively fast voltage ramps can elicit a mixed persistent and fast current, whose amplitude depends on the kinetics of fast inactivation, and very slow voltage ramps can induce inactivation of persistent current. Modified from Ref. with permission from Neuropharmacology. D: the resurgent sodium current is generated in some cell types during repolarizations from positive voltages to moderately negative potentials, and gain-of-function mutations can increase it, whereas loss-of-function mutations can decrease it. Modified from Ref. with permission from Journal of Neuroscience. E: loss-of-function mutations of sodium channels decrease neuronal excitability. a: Loss of action potential firing in GABAergic neurons from Scn1a-knockout mice. Action potential discharges, recorded in current-clamp mode with the whole cell configuration of the patch-clamp technique, elicited by injecting depolarizing currents of increasing amplitude in control (+/+), heterozygote Scn1a^+/− (+/−), and homozygote Scn1a^−/− (−/−) GABAergic neurons. b: Input-output relationships of the number of action potentials vs. the injected current show a large reduction of excitability in Scn1a^−/− neurons and less severe reduction in Scn1a^+/− neurons. The rheobase (i.e., the minimum depolarizing current that elicits an action potential) is not modified in this model, but loss of function of some sodium channels can increase it. c: Representative single action potentials, recorded for each genotype of GABAergic neurons and elicited by the same injected current amplitude (35 pA), show reduced amplitude with Na_V1.1 loss of function, as well as increased width and half-width, which are defined as the width at the base and at the half-maximum amplitude of the action potential (AP). d: Modified from Ref. with permission from Nature Neuroscience. F: action potential discharges recorded in cultured GABAergic neurons transfected with wild-type (WT) Na_V1.1 or the familial hemiplegic migraine gain-of-function L1649Q mutant show increased excitability when the mutant is expressed, as quantified in the input-output relationships (bottom). Modified from Ref. with permission from Proceedings of the National Academy of Sciences of the United States of America. Sodium channel gain of function can also decrease rheobase and modify features of action potentials (e.g., amplitude, slope, width); not shown.

FIGURE 3.

Spectrum of mutations and phenotypes for SCN4A/Na_V1.4. A: phenotypic spectrum. Most SCN4A mutations cause Na_V1.4 gain of function with different mechanisms, including induction of gating pore current, leading to specific clinical entities. A minority of mutations cause loss of function, inducing clinical symptoms when the loss is >50% (homozygosis or compound heterozygosis). The few patients with complete loss-of-function mutations in homozygosis showed perinatal lethality. B: molecular map of SCN4A mutations color-coded as indicated: hyperkalemic periodic paralysis (HyperPP), hypokalemic periodic paralysis (HypoPP), normokalemic periodic paralysis (NormoPP), paramyotonia congenita (PMC), potassium-aggravated myotonia (PAM), cold-aggravated myotonia (CAM), congenital myopathy (SCM), and congenital myasthenic syndrome (CMS).

FIGURE 4.

Structural features of Nav1.4 mutations. A–D: structural location of periodic paralysis mutations. CMS, congenital myasthenic syndrome; GEFS + 1, generalized epilepsy with febrile seizures plus 1; HOKPP2, hypokalemic periodic paralysis; HYPP, hyperkalemic periodic paralysis; MYOSCN4A, myotonia caused by SCN4A mutations; NKPP, normokalemic periodic paralysis; PMC, paramyotonia congenita; VSD, voltage-sensing domain. Adapted from Ref. with permission from Science.

FIGURE 5.

Pathogenic gating pore current and structure. A, left: central pore Na⁺ currents (inset) and conductance (G)/voltage (V) curve for Na_vAb/R2S recorded during 200-ms depolarizations from −200 mV to the indicated potentials. Center: leak Na⁺ currents for wild-type (WT; black) and Na_vAb/R2S (blue). Note the larger negative leak currents in Na_vAb/R2S due to gating pore current. Right: current (I_gp)/V curves for nonlinear leak currents for Na_vAb/R2S (blue) or Na_vAb/WT (black) elicited by depolarization from −100 mV to the indicated potentials. B, left: central pore Na⁺ currents (inset) and G/V curve for Na_vAb/R3G from a holding potential of −160 mV (filled circles). Voltage dependence of steady-state inactivation (open circles) for Na_vAb/R3G. Center: leak Na⁺ currents for Na_VAb/R3G (red) or Na_VAb/WT (black) for voltage steps from 0 mV to the indicated potentials. Right: I_gp/V curves for nonlinear leak currents for Na_vAb/R3G (red) or Na_VAb/WT (black, n = 11). Note the larger positive leak currents in Na_vAb/R3G due to gating pore current. C: structure of a pathogenic gating pore (GP) in a normokalemic periodic paralysis (NormoPP) mutation. Magenta shading, water-accessible space determined by MOLE2. HCS, hydrophobic constriction site; R1–R4, gating charges; S1–S4, transmembrane segments. Adapted from Ref. with permission from Nature.

FIGURE 6.

Spectrum of mutations and phenotypes for SCN1A/Na_V1.1. A: phenotypic spectrum of SCN1A mutations. Most SCN1A mutations cause epileptic phenotypes, including the severe developmental and epileptic encephalopathy Dravet syndrome and the milder form genetic epilepsy with febrile seizures plus (GEFS+), which, however, shows large phenotypic variability and can include severe cases. SCN1A variants may be also involved in febrile seizure phenotypes (FS) that can include development of temporal lobe epilepsy with hippocampal sclerosis. These forms are caused by loss of function of Na_V1.1 in heterozygosis, with often complete loss of function for Dravet syndrome, which is modeled by Scn1a^+/− knockout mice. Gain of function of Na_V1.1 has been identified for hemiplegic migraine (FHM3) mutations and proposed for mutations that cause an extremely severe early infantile epileptic encephalopathy (EIEE), although functional studies for this form have been performed for a single mutation. P15, postnatal day 15. B: molecular map of the Na_V1.1 sodium channel with the location of hemiplegic migraine mutations color-coded as indicated (ERDB, elicited repetitive daily blindness). C: molecular map of the Na_V1.1 sodium channel with the location of SCN1A EIEE mutations. Mutations causing other phenotypes are not shown because there are too many for a graphical representation.

FIGURE 7.

Cellular and systems phenotypes of Dravet syndrome (DS) in mice. A: electroencephalogram of a generalized tonic-clonic seizure in a DS mouse. B: loss of firing in interneurons. APs, action potentials; pA, picoamperes of stimulating current. Left: parvalbumin-expressing interneurons in layer V of the cerebral cortex. Black, wild type; blue, DS. Right, somatostatin-expressing interneurons in layer V of the cerebral cortex. Black, wild type (WT); gold, DS. Adapted from Ref. with permission from Proceedings of the National Academy of Sciences of the United States of America. C: thermal induction of seizures. P, postnatal day. D: autistic-like social interaction behavior. E, empty; C, center; M, mouse. E: context-dependent fear conditioning. #P < 0.05. Adapted from Ref. with permission from Nature.

FIGURE 8.

Synergistic interactions between SCN1A mutations and seizures leading to pathological remodeling and development of severe phenotypes. Top: protocol timeline. Bottom: outcomes of the experiments that showed that the Scn1a^R1648H/+ knockin mice in the 129-C57BL/6 genetic background have an asymptomatic phenotype. The induction of short repeated seizures had no effect on wild-type (WT) mice, but it transformed the asymptomatic phenotype of Scn1a^R1648H/+ mice into a severe Dravet syndrome-like phenotype, including frequent spontaneous seizures and cognitive/behavioral deficits. In these mice, there were no major modifications in cytoarchitecture or neuronal death but increased excitability of hippocampal dentate gyrus granule cells and increased expression of calbindin, consistent with a pathological remodeling of neuronal functions. Thus, an SCN1A mutation is a prerequisite for a long-term deleterious effect of seizures on the brain, indicating a clear interaction between seizures and the mutation (2-hit) for the development of a severe phenotype generated by pathological remodeling. ECoG, electrocorticogram. Adapted from Ref. with permission from Neurobiology of Disease.

FIGURE 9.

Rescue of expression and function of SCN1A/Na_V1.1 mutants. A: mutations of Na_V1.1 and protein folding. Left: folding-defective mutants are recognized as incorrectly folded proteins by the quality control system of the endoplasmic reticulum (ER) and degraded. Center: folding defects can be rescued by molecular interactions with interacting proteins or pharmacological chaperones, which probably act by stabilizing a correct folding conformation and preventing degradation. They also interact with rescued proteins by modifying their functional properties (purple arrow), which can be a major drawback for therapeutic applications of pharmacological chaperones. Rescue of epileptogenic Na_V1.1 folding-defective mutants attenuates the loss of function but, different than for FHM mutations, never induces gain of function. Right: engineered peptide toxins targeted to the ER can rescue Na_V1.1 folding-defective mutants but, unlike pharmacological chaperones, do not interact with rescued proteins at the plasma membrane. B, left: the cDNA of Centruroides sculpturatus Ewing (CsEI) β-scorpion toxin, sequence-optimized for expression in human cells, was engineered to target the peptide to the ER by insertion of an NH₂-terminal calreticulin ER targeting sequence (orange) and a COOH-terminal KDEL ER retention motif (red); cells transfected with a plasmid containing the engineered cDNA expressed the engineered ER-resident toxin that rescued some Na_V1.1 mutants and was not released extracellularly, as tested with immunoblots of the extracellular medium and with functional evaluations (228). Right: interaction of the toxin with sodium channels at neurotoxin receptor site 4 in domain II; toxin binding sites are color coded as indicated. Adapted from Ref. with permission of Neurobiology of Disease and from Ref. with permission from Biochimie.

FIGURE 10.

Comparison of proposed mechanisms for generation of migraine attacks by gain-of-function Na_V1.1 FHM3 mutations and seizure generation by Na_V1.1 epileptogenic mutations. Left: in migraine the genetic background is important for determining an intrinsic threshold for migraine attacks, which is modulated by internal and external factors (trigger stimuli). Emotional stress and minor head trauma are among the most common triggers of hemiplegic migraine attacks (250, 251). Some triggers are thought to induce excessive neuronal firing and consequently lead to extracellular K⁺ accumulation, which can eventually lead to long-lasting neuronal depolarization and silencing of firing caused by inactivation of sodium channels (depolarization block). These events produce cortical spreading depolarization (CSD), a wave of transient network hyperexcitability leading to a long-lasting depolarization block of neuronal firing. CSD directly causes aura, and it could also induce headache by activating trigeminal nociceptors and hemiparesis (248, 271). FHM3 Na_V1.1 gain-of-function mutations can lower the triggering threshold of migraine attacks by increasing excitability of GABAergic neurons, which can induce spike-dependent accumulation of extracellular potassium that engages the spiking of glutamatergic neurons, eventually leading to depolarizing block and CSD initiation. Extracellular accumulation of the excitatory neurotransmitter glutamate plays a minor role in this proposed GABAergic neuron-dependent mechanism (272). Right: similar to migraine, it is hypothesized that epileptogenic mutations lower the threshold for seizure generation, although seizure triggers are less clearly identified than migraine triggers (248). Epileptogenic Na_V1.1 loss-of-function mutations lower seizure threshold by reducing firing of GABAergic neurons, which leads to reduced GABAergic synaptic transmission and reduced inhibition in neuronal circuits, which can lead to generation of epileptic activity. Comorbidities could be generated by both seizures and the direct effect of the mutation (140, 141, 172).

FIGURE 11.

Spectrum of mutations and phenotypes for SCN2A/Na_V1.2. A: SCN2A/Na_V1.2 mutations inducing mild gain of function cause benign neonatal-infantile familial seizures (BNIFS) with onset between 3 and 6 mo of age. Neonatal-early infantile developmental and epileptic encephalopathies (NEIDEE) with onset before 3 mo are in general caused by mutations that induce larger gain of function. Mutations inducing loss of function cause infantile-childhood developmental and epileptic encephalopathies (ICDEE), with onset after 3 mo. Complete loss of function in heterozygosis (haploinsufficiency) can lead to behavioral/cognitive phenotypes without epilepsy: autism spectrum disorders (ASD), intellectual disability, or schizophrenia. Haploinsufficiency in Scn2a^+/− mice causes a relatively mild and age-dependent phenotype including short absence-like seizures, autistic/schizophrenic traits, and memory dysfunctions. Homozygote Scn2a^−/− knockout (KO) mice show embryonic mortality. B: molecular map of the Na_V1.2 sodium channel with the location of 19 missense SCN2A mutations in BNIFS (yellow) and 49 missense SCN2A mutations in developmental and epileptic encephalopathy (DEE: both ICDEE and NEIDEE) (green). C: molecular map of the Na_V1.2 sodium channel with the location of 12 missense (red) and 9 protein-truncating (blue) SCN2A variants identified in ASD cases.

FIGURE 12.

Phenotypes of heterozygous Scn2a knockout (Scn2a^+/−) mice. A: absence-like seizures with spike and wave discharges (SWDs) associated with electromyogram (EMG) suppression are observed in 10- to 27-wk-old Scn2a^+/− mice, as shown by electrocorticogram and multisite local field potential (ECoG-LFP) recordings, which reveal the predominant appearance of LFP epileptiform discharges in medial prefrontal cortex (mPFC) and caudate putamen (CPu). Black arrowheads indicate the onset of SWD. Scale bars: vertical, 500 µV; horizontal, 1 s. Modified from Ref. with permission from Communications Biology. B: stereotyped behaviors consistent with autistic-like traits are present in young (a–c) but not adult (d–f) Scn2a^+/− (HZ) mice. WT, wild type. a and d: Time spent in self-grooming. b and e: Number of marbles buried. c and f: Persistence in repetitive rotarod trials, which evaluate repetitive motor behaviors. These stereotyped behaviors are not observed in adult [postnatal day (P)60–P95] mice. Modified from Ref. with permission from Scientific Reports. C: impaired somatic excitability. a: Severe impairment in developing cortical pyramidal neurons from P4–P7 Scn2a^+/− mice. Morphology of a developing pyramidal cell (left) and action potential discharges (right). b: Moderate impairment in mature neurons from adult Scn2a^+/− mice (>P60). Morphology of a mature, thick-tufted pyramidal cell (left) and action potential discharges (right). Modified from Ref. with permission from Neuron. D: backpropagating dendritic excitability is impaired in mature cortical layer V pyramidal neurons of Scn2a^+/− mice. Calcium transients evoked by trains of action potential duplets were recorded at various locations throughout the apical dendrite. In WT neurons duplets reliably evoke calcium transients throughout the apical dendrite, whereas calcium transients in Scn2a^+/− neurons rapidly diminish in amplitude with increasing distance from the soma, becoming virtually absent in the most distal dendritic branches. G/G_sat, relative fluorescence intensity; V_m, membrane voltage; I_inj, injected current. Modified from Ref. with permission from Neuron. E: decreased long-term potentiation (LTP) induced by theta burst stimulation (10 trains of 4 pulses at 100 Hz) at hippocampal Schaffer collaterals-CA1 synapses of 3-mo-old floxed Scn2a^+/− mice (HT; red) compared with WT littermates. fEPSP, field excitatory postsynaptic potential. Modified from Ref. with permission from Frontiers in Molecular Neuroscience.

FIGURE 13.

Spectrum of mutations and phenotypes for SCN8A/Na_V1.6. A: phenotypic spectrum. Gain-of-function SCN8A/Na_V1.6 mutations can cause relatively mild epilepsy [e.g., benign familial infantile seizures (BFIS)], paroxysmal dyskinesia when the functional effect is moderate, or severe developmental and epileptic encephalopathy (DEE13) when the functional effect is stronger. Loss-of-function mutations can cause intellectual disability (ID), autism [autism spectrum disorders (ASD)], or motor dysfunctions without epilepsy, which can be caused also by mutations that cause a massive gain of function inducing loss of neuronal firing by depolarizing block. Knockin mice carrying gain-of-function mutations show hyperexcitability of excitatory neurons and phenotype consistent with DEE13. Knockout (KO) mice reproducing complete loss of function in heterozygosis (haploinsufficiency) show anxiety and absence seizures. Mice with complete loss of function in homozygosis show severe motor dysfunctions and juvenile lethality. B: molecular map of the Na_V1.6 sodium channel with the location of SCN8A mutations color-coded as indicated.

FIGURE 14.

Spectrum of mutations and phenotypes in SCN3A/Na_V1.3. A: phenotypic spectrum of Na_V1.3 mutations. Most SCN3A/Na_V1.3 pathogenic mutations cause gain of function, with milder ones identified in patients with focal epilepsy and stronger ones in patients with early infantile epileptic encephalopathy often including extended polymicrogyria (DEE62) or speech/oral motor dysfunctions and polymicrogyria limited to the perisylvian cerebral cortex without epilepsy. Loss-of-function mutations have been identified in 2 patients with DEE62 and 1 patient with developmental delay/autism spectrum disorders (ASD), but it is not clear how they can give rise to phenotypes that are similar to those of the gain-of-function mutations, which are the large majority. Haploinsufficiency in Scn3a^+/- knockout (KO) mice causes deficits in locomotor activity and increased susceptibility to convulsants but not an overt epileptic phenotype or behavioral dysfunctions. B: molecular map of the Na_V1.3 sodium channel with the location of SCN3A mutations color-coded as indicated.