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.     

Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.     

Differential expression of sodium channel mRNAs in rat peripheral nervous system and innervated tissues

RNA blot hybridization analyses using probes specific for sodium channels I, II and III revealed high levels of sodium channel I mRNA and low levels of sodium channel II and III mRNAs in peripheral nervous system (PNS) tissues. The developmental expression patterns of these mRNAs were generally similar to those described for the central nervous system. The small amounts of sodium channel I and III mRNAs present in tongue muscle were greatly reduced after partial denervation. Expression of the three sodium channels thus appears to be restricted to the nervous system. Putative novel additional mRNAs, specifically expressed in the PNS, were detected with a probe that recognizes nucleotide sequences common to sodium channels I, II and III.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Electrical activity, cAMP, and cytosolic calcium regulate mRNA encoding sodium channel alpha subunits in rat muscle cells

The number of sodium channels increases sharply during development of rat skeletal muscle cells in vitro. An 8.5 kb mRNA encoding sodium channel alpha subunit rises to a peak on day 13 in vitro and falls to a value of 50% of the peak by day 18, consistent with the conclusion that mRNA abundance is a major determinant of the rapid rise in sodium channel number. Electrical activity and increased cytosolic calcium decrease the level of alpha subunit mRNA, and cAMP increases its level in parallel with changes in the number of sodium channels. The similarity between the changes in mRNA levels and sodium channel density indicates that the regulation of alpha subunit mRNA level is an important mechanism of feedback regulation of sodium channel density by electrical activity in developing rat muscle cells.     

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

Excitability constraints on voltage-gated sodium channels

We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% identical by sequence. This sequence similarity presumably corresponds to similarity of function, consistent with the idea that these properties must be tightly constrained. However, the multiplicity of genes encoding sodium channels raises the question: why are there so many? We demonstrate that the simplest theoretical constraints bounding sodium channel diversity--the requirements of membrane excitability and the uniqueness of the resting potential--act directly on constraining sodium channel properties. We compare the predicted constraints with functional data on mammalian sodium channel properties collected from the literature, including 172 different sets of measurements from 40 publications, wild-type and mutant, under a variety of conditions. The data from all channel types, including mutants, obeys the excitability constraint; on the other hand, channels expressed in muscle tend to obey the constraint of a unique resting potential, while channels expressed in neuronal tissue do not. The excitability properties alone distinguish the nine sodium channels into four different groups that are consistent with phylogenetic analysis. Our calculations suggest interpretations for the functional differences between these groups.     

Primary structure of squid sodium channel deduced from the complementary DNA sequence.

The complete amino acid sequence of a sodium channel from squid Loligo bleekeri has been deduced by cloning and sequence analysis of the complementary DNA. The deduced sequence revealed an organization virtually identical to the vertebrate sodium channel proteins; four homologous domains containing all six membrane-spanning structures are repeated in tandem with connecting linkers of various sizes. A unique feature of the squid Na channel is the 1,522 residue sequence, approximately three fourths of those of the rat sodium channels I, II and III.     

Correlating physiology with gene expression in striatal cholinergic neurones

The expression of 34 transmitter-related genes has been examined in the cholinergic neurones of rat striatal brain slices, with the aim of correlating gene expression with functional activity. The mRNAs encoding types I, II/IIA, and III alpha subunits of the voltage-sensitive sodium channels were detected, suggesting the presence of these three types of sodium channel. Similarly, mRNAs encoding all four alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptor subunits and the NR1 and NR2A, 2B, and 2D subunits of the NMDA-type glutamate receptors were detected, suggesting that various combinations of these subunits mediate the cellular response to synaptically released glutamate. Other mRNAs detected included the NK1 and NK3 tachykinin receptors, all four known adenosine receptors, and the GABA-synthesising enzyme glutamate decarboxylase. Subpopulations of these cholinergic neurones have been identified on the basis of the expression of the NK3 tachykinin receptor in 5% and the trkC neurotrophin receptor in 12% of the cells investigated.     

Structure of a putative sodium channel from the sea anemoneAiptasia pallida

A cDNA encoding a full length putative sodium channel has been cloned from the sea anemoneAiptasia pallida. The deduced protein, named AiNal, has a predicted molecular weight of 205 000 Da. It shows high structural similarity to other sodium channels from both invertebrates and vertebrates, and its structure is consistent with the four domain, six transmembrane segment motif of all known voltage-gated sodium channels. In the region purported to constitute the tetrodotoxin (TTX) receptor of sodium channels, AiNal differs from the TTX-sensitive motif, suggesting that currents carried by this channel would be insensitive to TTX. The presence of a conventional sodium channel protein in anemones indicates, for the first time, that neurons in sea anemones are likely to be capable of producing fast, overshooting action, potentials.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Interaction of scorpion alpha-toxins with cardiac sodium channels: binding properties and enhancement of slow inactivation

The effects of the scorpion alpha-toxins Lqh II, Lqh III, and LqhalphaIT on human cardiac sodium channels (hH1), which were expressed in human embryonic kidney (HEK) 293 cells, were investigated. The toxins removed fast inactivation with EC(50) values of <2.5 nM (Lqh III), 12 nM (Lqh II), and 33 nM (LqhalphaIT). Association and dissociation rates of Lqh III were much slower than those of Lqh II and LqhalphaIT, such that Lqh III would not dissociate from the channel during a cardiac activation potential. The voltage dependence of toxin dissociation from hH1 channels was nearly the same for all toxins tested, but it was different from that found for skeletal muscle sodium channels (muI; Chen et al. 2000). These results indicate that the voltage dependence of toxin binding is a property of the channel protein. Toxin dissociation remained voltage dependent even at high voltages where activation and fast inactivation is saturated, indicating that the voltage dependence originates from other sources. Slow inactivation of hH1 and muI channels was significantly enhanced by Lqh II and Lqh III. The half-maximal voltage of steady-state slow inactivation was shifted to negative values, the voltage dependence was increased, and, in particular for hH1, slow inactivation at high voltages became more complete. This effect exceeded an expected augmentation of slow inactivation owing to the loss of fast inactivation and, therefore, shows that slow sodium channel inactivation may be directly modulated by scorpion alpha-toxins.     

Sodium Channel Fragments: Contributions to Voltage Sensitivity and Ion Selectivity

The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel. Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichroism spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader with repeat III. These structural and functional correlates suggest alternative gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-recognition between the four P-regions is a key component of intact sodium channel selectivity. In addition, the P-region from domain III is significantly voltage-sensitive and molecular dynamics simulations show that the C-terminal part of P-regions is mainly helical whilst the N-terminus tends to unfold. Such specializations of the four domains both in gating and selectivity are independently confirmed in recent electrophysiological studies.     

A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr¹⁴⁹⁴ and Tyr¹⁴⁹⁵) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.     

Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C

Neurotransmitters modulate sodium channel availability through activation of G protein-coupled receptors, cAMP-dependent protein kinase (PKA), and protein kinase C (PKC). Voltage-dependent slow inactivation also controls sodium channel availability, synaptic integration, and neuronal firing. Here we show by analysis of sodium channel mutants that neuromodulation via PKA and PKC enhances intrinsic slow inactivation of sodium channels, making them unavailable for activation. Mutations in the S6 segment in domain III (N1466A,D) either enhance or block slow inactivation, implicating S6 segments in the molecular pathway for slow inactivation. Modulation of N1466A channels by PKC or PKA is increased, whereas modulation of N1466D is nearly completely blocked. These results demonstrate that neuromodulation by PKA and PKC is caused by their enhancement of intrinsic slow inactivation gating. Modulation of slow inactivation by neurotransmitters acting through G protein-coupled receptors, PKA, and PKC is a flexible mechanism of cellular plasticity controlling the firing behavior of central neurons.     

Genomic Organization of the Human Skeletal Muscle Sodium Channel Gene

Voltage-dependent sodium channels are essential for normal membrane excitability and contractility in adult skeletal muscle. The gene encoding the principal sodium channel α-subunit isoform in human skeletal muscle (SCN4A) has recently been shown to harbor point mutations in certain hereditary forms of periodic paralysis. We have carried out an analysis of the detailed structure of this gene including delineation of intron-exon boundaries by genomic DNA cloning and sequence analysis. The complete coding region of SCN4A is found in 32.5 kb of genomic DNA and consists of 24 exons (54 to > 2.2 kb) and 23 introns (97 bp-4.85 kb). The exon organization of the gene shows no relationship to the predicted functional domains of the channel protein and splice junctions interrupt many of the transmembrane segments. The genomic organization of sodium channels may have been partially conserved during evolution as evidenced by the observation that 10 of the 24 splice junctions in SCN4A are positioned in homologous locations in a putative sodium channel gene in Drosophila (para). The information presented here should be extremely useful both for further identifying sodium channel mutations and for gaining a better understanding of sodium channel evolution.     

Real-time neuronal homeostasis by coordinating VGSC intrinsic properties

Homeostasis of internal environment and cellular metabolism ensures cells’ functions to be stable in living organisms. Cellular homeostasis is believed to be maintained via feedback or feedforward manners. We report a novel mechanism that maintains neuronal homeostasis through coordinating the intrinsic properties of single molecules concurrently. Spike encoding and sodium channel dynamics at cortical neurons were studied by patch-clamp recording. Voltage-gated sodium channels set refractory period and threshold potential toward different directions to stabilize the energetic barrier for firing sequential action potentials. This neuronal homeostasis is not affected by intracellular Ca²⁺ signals and membrane potentials. Real-time homeostasis maintains precise and reliable neuronal encoding without any destabilization.