Identification of a novel human voltage-gated sodium channel alpha subunit gene, SCN12A

We have cloned a cDNA encoding a novel human voltage-gated sodium channel alpha subunit gene, SCN12A, from human brain. Two alternative splicing variants for SCN12A have been identified. The longest open reading frame of SCN12A encodes 1791 amino acid residues. The deduced amino acid sequence of SCN12A shows 37-73% similarity with various other mammalian sodium channels. The presence of a serine residue (S360) in the SS2 segment of domain I suggests that SCN12A is resistant to tetrodotoxin (TTX), as in the cases of rat Scn10a (rPN3/SNS) and rat Scn11a (NaN/SNS2). SCN12A is expressed predominantly in olfactory bulb, hippocampus, cerebellar cortex, spinal cord, spleen, small intestine, and placenta. Although expression level could not be determined, SCN12A is also expressed in dorsal root ganglia (DRG). Both neurons and glial cells express SCN12A. SCN12A maps to human chromosome 3p23-p21.3. These results suggest that SCN12A is a tetrodotoxin-resistant (TTX-R) sodium channel expressed in the central nervous system and nonneural tissues.     

Cloning and expression study of the mouse tetrodotoxin-resistant voltage-gated sodium channel alpha subunit NaT/Scn11a

We have cloned a tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel alpha subunit from a mouse cDNA library and designated it as NaT. It encodes 1765 amino acid residues and is virtually identical to that of Scn11a, which has been reported recently, except for 40 nt and 14 aa substitutions. The amino acid identity of NaT/Scn11a with rat NaN/SNS2 is 88%. NaT/Scn11a was mapped to mouse chromosome 9F3-F4 by fluorescence in situ hybridization (FISH). While rat NaN/SNS2 has been reported to be expressed specifically in the peripheral sensory neurons, NaT/Scn11a is expressed not only in the peripheral sensory neurons but also in the spinal cord, uterus, testis, ovary, placenta, and small intestine. NaT is detectable in mouse embryos 15 days postcoitus (p.c.), around the phase of organogenesis and gonadal differentiation. These findings demonstrate a unique distribution of NaT/Scn11a and suggest some of its roles in the above-mentioned processes.     

Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons

Two tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels, SNS and NaN, are preferentially expressed in small dorsal root ganglia (DRG) and trigeminal ganglia neurons, most of which are nociceptive, of rat and mouse. We report here the sequence of NaN from human DRG, and demonstrate the presence of two TTX-R currents in human DRG neurons. One current has physiological properties similar to those reported for SNS, while the other displays hyperpolarized voltage-dependence and persistent kinetics; a similar TTX-R current was recently identified in DRG neurons of sns-null mouse. Thus SNS and NaN channels appear to produce different currents in human DRG neurons.     

Three brain sodium channel alpha-subunit genes are clustered on the proximal segment of mouse chromosome 2.

We have used long-range physical mapping and restriction fragment length polymorphisms between two mouse species to determine the chromosomal organization and location of the genes encoding three distinct isoforms of the alpha-subunit of the brain sodium channel. Physical mapping by pulsed-field gel electrophoresis has established that Scn2a and Scn3a (genes encoding type II and type III sodium channel alpha-subunit isoforms) are physically linked and are separated by a maximum distance of 600 kb. The segregation of restriction fragment length variations in backcross progeny of a Mus musculus and Mus spretus mating indicates that Scn 1 a (gene encoding the type I sodium channel alpha subunit) and Scn2a are tightly linked and are separated by a distance of 0.7 cM. Linkage analysis in backcross and recombinant inbred (BXD and AKXD) strains of mice localized the three sodium channel genes to the proximal segment of mouse chromosome 2 and suggested the probable gene order centromere-Hc-Neb-Pmv7-Scn2a/Scn3a-Scn1a-Mpmv 14. These results indicate that the three isoforms of the brain sodium channel alpha-subunit are encoded by three distinct genes that share a common ancestral origin.     

Altered Function of the SCN1A Voltage-gated Sodium Channel Leads to ��-Aminobutyric Acid-ergic (GABAergic) Interneuron Abnormalities

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),⁷ severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a^RH/RH mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a^RH/+ heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a^RH/+ and Scn1a^RH/RH mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.     

Gain-of-Function Mutations in SCN11A Cause Familial Episodic Pain

Many ion channel genes have been associated with human genetic pain disorders. Here we report two large Chinese families with autosomal-dominant episodic pain. We performed a genome-wide linkage scan with microsatellite markers after excluding mutations in three known genes (SCN9A, SCN10A, and TRPA1) that cause similar pain syndrome to our findings, and we mapped the genetic locus to a 7.81 Mb region on chromosome 3p22.3–p21.32. By using whole-exome sequencing followed by conventional Sanger sequencing, we identified two missense mutations in the gene encoding voltage-gated sodium channel Na_v1.9 (SCN11A): c.673C>T (p.Arg225Cys) and c.2423C>G (p.Ala808Gly) (one in each family). Each mutation showed a perfect cosegregation with the pain phenotype in the corresponding family, and neither of them was detected in 1,021 normal individuals. Both missense mutations were predicted to change a highly conserved amino acid residue of the human Na_v1.9 channel. We expressed the two SCN11A mutants in mouse dorsal root ganglion (DRG) neurons and showed that both mutations enhanced the channel’s electrical activities and induced hyperexcitablity of DRG neurons. Taken together, our results suggest that gain-of-function mutations in SCN11A can be causative of an autosomal-dominant episodic pain disorder.     

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).     

Genetic modifiers affecting severity of epilepsy caused by mutation of sodium channelScn2a

Mutations in the voltage-gated sodium channels SCN1A and SCN2A are responsible for several types of human epilepsy. Variable expressivity among family members is a common feature of these inherited epilepsies, suggesting that genetic modifiers may influence the clinical manifestation of epilepsy. The transgenic mouse model Scn2a^Q54 has an epilepsy phenotype as a result of a mutation in Scn2a that slows channel inactivation. The mice display progressive epilepsy that begins with short-duration partial seizures that appear to originate in the hippocampus. The partial seizures become more frequent and of longer duration with age and often induce secondary generalized seizures. Clinical severity of the Scn2a^Q54 phenotype is influenced by genetic background. Congenic C57BL/6J.Q54 mice exhibit decreased incidence of spontaneous seizures, delayed seizure onset, and longer survival in comparison with [C57BL/6J × SJL/J]F₁.Q54 mice. This observation indicates that strain SJL/J carries dominant modifier alleles at one or more loci that determine the severity of the epilepsy phenotype. Genome-wide interval mapping in an N₂ backcross revealed two modifier loci on Chromosomes 11 and 19 that influence the clinical severity of of this sodium channel-induced epilepsy. Modifier genes affecting clinical severity in the Scn2a^Q54 mouse model may contribute to the variable expressivity seen in epilepsy patients with sodium channel mutations.     

Functional Upregulation of Nav1.8 Sodium Channels on the Membrane of Dorsal Root Ganglia Neurons Contributes to the Development of Cancer-Induced Bone Pain

We have previously reported that enhanced excitability of dorsal root ganglia (DRG) neurons contributes to the development of bone cancer pain, which severely decreases the quality of life of cancer patients. Nav1.8, a tetrodotoxin-resistant (TTX-R) sodium channel, contributes most of the sodium current underlying the action potential upstroke and accounts for most of the current in later spikes in a train. We speculate that the Nav1.8 sodium channel is a potential candidate responsible for the enhanced excitability of DRG neurons in rats with bone cancer pain. Here, using electrophysiology, Western blot and behavior assays, we documented that the current density of TTX-R sodium channels, especially the Nav1.8 channel, increased significantly in DRG neurons of rats with cancer-induced bone pain. This increase may be due to an increased expression of Nav1.8 on the membrane of DRG neurons. Accordantly, blockade of Nav1.8 sodium channels by its selective blocker A-803467 significantly alleviated the cancer-induced mechanical allodynia and thermal hyperalgesia in rats. Taken together, these results suggest that functional upregulation of Nav1.8 channels on the membrane of DRG neurons contributes to the development of cancer-induced bone pain.     

Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane

The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.     

The α-subunit of the skeletal muscle sodium channel is encoded proximal to Tk-1 on mouse Chromosome 11

Recent evidence suggests that the human neuromuscular disorders, hyperkalemic periodic paralysis and paramyotonia congenita, are both caused by genetic defects in the -subunit of the adult skeletal muscle sodium channel, which maps near the growth hormone cluster (GH) on Chromosome (Chr) 17q. In view of the extensive homology between this human chromosome and mouse Chr 11, we typed an interspecies backcross to determine whether the murine homolog (Scn4a) of this sodium channel gene mapped within the conserved chromosomal segment. The cytosolic thymidine kinase gene, Tk-1, was also positioned on the genetic map of Chr 11. Both Scn4a and Tk-1 showed clear linkage to mouse Chr 11 loci previously typed in this backcross, yielding the map order: Tr ^J-(Re, Hox-2, Krt-1)-Scn4a-Tk-1. No mouse mutant that could be considered a model of either hyperkalemic periodic paralysis or paramyotonia congenita has been mapped to the appropriate region of mouse Chr 11. These data incorporate an additional locus into the already considerable degree of homology observed for these human and mouse chromosomes. These data are also consistent with the view that the conserved segment region may extend to the telomere on mouse Chr 11 and on human 17q.     

Mapping genetic modifiers of survival in a mouse model of Dravet syndrome

Epilepsy is a common neurological disorder affecting approximately 1% of the population. Mutations in voltage‐gated sodium channels are responsible for several monogenic epilepsy syndromes. More than 800 mutations in the voltage‐gated sodium channel SCN1A have been reported in patients with generalized epilepsy with febrile seizures plus and Dravet syndrome. Heterozygous loss‐of‐function mutations in SCN1A result in Dravet syndrome, a severe infant‐onset epileptic encephalopathy characterized by intractable seizures, developmental delays and increased mortality. A common feature of monogenic epilepsies is variable expressivity among individuals with the same mutation, suggesting that genetic modifiers may influence clinical severity. Mice with heterozygous deletion of Scn1a (Scn1a^+/?) model a number of Dravet syndrome features, including spontaneous seizures and premature lethality. Phenotype severity in Scn1a^+/? mice is strongly dependent on strain background. On the 129S6/SvEvTac strain Scn1a^+/? mice exhibit no overt phenotype, whereas on the (C57BL/6J × 129S6/SvEvTac)F1 strain Scn1a^+/? mice exhibit spontaneous seizures and early lethality. To systematically identify loci that influence premature lethality in Scn1a^+/? mice, we performed genome scans on reciprocal backcrosses. Quantitative trait locus mapping revealed modifier loci on mouse chromosomes 5, 7, 8 and 11. RNA‐seq analysis of strain‐dependent gene expression, regulation and coding sequence variation provided a list of potential functional candidate genes at each locus. Identification of modifier genes that influence survival in Scn1a^+/? mice will improve our understanding of the pathophysiology of Dravet syndrome and may suggest novel therapeutic strategies for improved treatment of human patients.