Association of rare missense variants in the second intracellular loop of NaV1.7 sodium channels with familial autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder often accompanied by intellectual disability, language impairment and medical co-morbidities. The heritability of autism is high and multiple genes have been implicated as causal. However, most of these genes have been identified in de novo cases. To further the understanding of familial autism, we performed whole-exome sequencing on five families in which second- and third-degree relatives were affected. By focusing on novel and protein-altering variants, we identified a small set of candidate genes. Among these, a novel private missense C1143F variant in the second intracellular loop of the voltage-gated sodium channel NaV1.7, encoded by the SCN9A gene, was identified in one family. Through electrophysiological analysis, we show that NaV1.7C1143F exhibits partial loss-of-function effects, resulting in slower recovery from inactivation and decreased excitability in cultured cortical neurons. Furthermore, for the same intracellular loop of NaV1.7, we found an excess of rare variants in a case-control variant-burden study. Functional analysis of one of these variants, M932L/V991L, also demonstrated reduced firing in cortical neurons. However, although this variant is rare in Caucasians, it is frequent in Latino population, suggesting that genetic background can alter its effects on phenotype. Although the involvement of the SCN1A and SCN2A genes encoding NaV1.1 and NaV1.2 channels in de novo ASD has previously been demonstrated, our study indicates the involvement of inherited SCN9A variants and partial loss-of-function of NaV1.7 channels in the etiology of rare familial ASD.

Influence of physical exercise on quality of life in individuals with spinal cord injury.

STUDY DESIGN: Retrospective cross-sectional study with anonymous postal data collection. OBJECTIVE: Regaining the best possible mobility and independence is not only the focus of the rehabilitation process for individuals with spinal cord injury (SCI), but also represents an important criterion for the individual's quality of life (QoL). Therefore, if and to what extent physical exercise (PE) influences the QoL of individuals with SCI was investigated. SETTING: The period of investigation extended from September 2007 to January 2008. Data were acquired from the BG Trauma Hospital Hamburg database and the German Wheelchair Sport Federation databases. METHODS: Analysis of 277 questionnaires of individuals with acquired SCI between the age of 16 and 65 years with complete wheelchair dependency in everyday life and lesion level lower C5. RESULTS: In all, 51.5% of all individuals were reported being actively involved in sports as opposed to 48.5% individuals not participating in sports. Individuals actively involved in sports have higher employment rate than physically inactive individuals with SCI. PE was identified as the main influencing determinant of QoL. This was particularly within the physical and psychological dimensions. CONCLUSION: In discovering the potential of individuals with SCI for getting involved in PE, the improvement of physical and coordinative skills with interaction between individuals with SCI and external sport groups should be an inherent part of the rehabilitation process. Individuals not having access to PE should be given the opportunity to participate in wheelchair mobility courses. This may improve the adherence to PE of individuals with SCI in post-clinical settings.

Identification of an intracellular peptide segment involved in sodium channel inactivation.

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.

Functional modulation of brain sodium channels by protein kinase C phosphorylation.

Voltage-gated sodium channels, which are responsible for the generation of action potentials in the brain, are phosphorylated by protein kinase C (PKC) in purified form. Activation of PKC decreases peak sodium current up to 80 percent and slows its inactivation for sodium channels in rat brain neurons and for rat brain type IIA sodium channel alpha subunits heterologously expressed in Chinese hamster ovary cells. These effects are specific for PKC because they can be blocked by specific peptide inhibitors of PKC and can be reproduced by direct application of PKC to the cytoplasmic surface of sodium channels in excised inside-out membrane patches. Modulation of brain sodium channels by PKC is likely to have important effects on signal transduction and synaptic transmission in the central nervous system.

Molecular determinants of state-dependent block of Na+ channels by local anesthetics.

Sodium ion (Na+) channels, which initiate the action potential in electrically excitable cells, are the molecular targets of local anesthetic drugs. Site-directed mutations in transmembrane segment S6 of domain IV of the Na+ channel alpha subunit from rat brain selectively modified drug binding to resting or to open and inactivated channels when expressed in Xenopus oocytes. Mutation F1764A, near the middle of this segment, decreased the affinity of open and inactivated channels to 1 percent of the wild-type value, resulting in almost complete abolition of both the use-dependence and voltage-dependence of drug block, whereas mutation N1769A increased the affinity of the resting channel 15-fold. Mutation I1760A created an access pathway for drug molecules to reach the receptor site from the extracellular side. The results define the location of the local anesthetic receptor site in the pore of the Na+ channel and identify molecular determinants of the state-dependent binding of local anesthetics.

A phosphorylation site in the Na+ channel required for modulation by protein kinase C.

Voltage-gated sodium channels are responsible for generation of action potentials in excitable cells. Activation of protein kinase C slows inactivation of sodium channels and reduces peak sodium currents. Phosphorylation of a single residue, serine 1506, that is located in the conserved intracellular loop between domains III and IV and is involved in inactivation of the sodium channel, is required for both modulatory effects. Mutant sodium channels lacking this phosphorylation site have normal functional properties in unstimulated cells but do not respond to activation of protein kinase C. Phosphorylation of this conserved site in sodium channel alpha subunits may regulate electrical activity in a wide range of excitable cells.

Functional properties of rat brain sodium channels expressed in a somatic cell line.

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.

Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase.

The function of voltage-gated sodium channels that are responsible for action potential generation in mammalian brain neurons is modulated by phosphorylation by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (cA-PK) and by protein kinase C (PKC). Reduction of peak sodium currents by cA-PK in intact cells required concurrent activation of PKC and was prevented by blocking phosphorylation of serine 1506, a site in the inactivation gate of the channel that is phosphorylated by PKC but not by cA-PK. Replacement of serine 1506 with negatively charged amino acids mimicked the effect of phosphorylation. Conversion of the consensus sequence surrounding serine 1506 to one more favorable for cA-PK enhanced modulation of sodium currents by cA-PK. Convergent modulation of sodium channels required phosphorylation of serine 1506 by PKC accompanied by phosphorylation of additional sites by cA-PK. This regulatory mechanism may serve to integrate neuronal signals mediated through these parallel signaling pathways.

Restoration of inactivation and block of open sodium channels by an inactivation gate peptide.

Inactivation of sodium channels terminates the sodium current responsible for initiation of action potentials in excitable cells. A hydrophobic sequence (isoleucine-phenylalanine-methionine, IFM), located in the inactivation gate segment connecting homologous domains III and IV of the sodium channel alpha subunit, is required for fast inactivation. A synthetic peptide containing the IFM sequence (acetyl-KIFMK-amide) restores fast inactivation to mutant sodium channels having a defective inactivation gate and to wild-type sodium channels having inactivation slowed by alpha-scorpion toxin. This peptide also competes with the intrinsic inactivation particle and binds to and blocks open sodium channels in a voltage- and frequency-dependent manner. A peptide (acetyl-KIQMK-amide) containing a mutation that prevents fast inactivation is not effective in restoring inactivation or in blocking open sodium channels. The results support the hypothesis that the sequence IFM serves as the inactivation particle of the sodium channel and suggest that it enters the intracellular mouth of the pore and occludes it during the process of inactivation.

Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line.

Type IIA rat brain Na+ channel alpha subunits were expressed in CHO cells by nuclear microinjection or by transfection using a vector containing both metallothionein and bacteriophage SP6 promoters. Stable cell lines expressing Na+ channels were isolated, and whole-cell Na+ currents of 0.9-14 nA were recorded. The mean level of whole-cell Na+ current (4.5 nA) corresponds to a cell surface density of approximately 2 channels active at the peak of the Na+ current per microns 2, a density comparable to that observed in the cell bodies of central neurons. The expressed Na+ channels had the voltage dependence, rapid activation and inactivation, and rapid recovery from inactivation characteristic of Na+ channels in brain neurons, bound toxins at neurotoxin receptor sites 1 and 3 with normal properties, and were posttranslationally processed to a normal mature size of 260 kd. Expression of Na+ channel cDNA in CHO cells driven by the metallothionein promoter accurately and efficiently reproduces native Na+ channel properties and provides a method for combined biochemical and physiological analysis of Na+ channel structure and function.

Persistent sodium currents through brain sodium channels induced by G protein betagamma subunits.

Persistent Na+ currents are thought to be important for integration of neuronal responses. Here, we show that betagamma subunits of G proteins can induce persistent Na+ currents. Coexpression of G beta2gamma3, G beta1gamma3, or G beta5gamma3, but not G beta1gamma1 subunits with rat brain type IIA Na+ channel alpha subunits in tsA-201 cells greatly enhances a component of Na+ current with a normal voltage dependence of activation but with dramatically slowed and incomplete inactivation and with steady-state inactivation shifted +37 mV. Synthetic peptides containing the proposed G betagamma-binding motif, Gln-X-X-Glu-Arg, from either adenylyl cyclase 2 or the Na+ channel alpha subunit C-terminal domain reversed the effect of G beta2gamma3 subunits. These results are consistent with direct binding of G betagamma subunits to the C-terminal domain of the Na+ channel, stabilizing a gating mode responsible for slowed and persistent Na+ current. Modulation of Na+ channel gating by G betagamma subunits is expected to have profound effects on neuronal excitability.

Functional modulation of brain sodium channels by cAMP-dependent phosphorylation.

Voltage-gated Na+ channels, which are responsible for the generation of action potentials in brain, are phosphorylated by cAMP-dependent protein kinase in vitro and in intact neurons. Phosphorylation by cAMP-dependent protein kinase reduces peak Na+ currents 40%--50% in membrane patches excised from rat brain neurons or from CHO cells expressing type IIA Na+ channels. Inhibition of basal cAMP-dependent protein kinase activity by transfection with a plasmid encoding a dominant negative mutant regulatory subunit increases Na+ channel number and activity, indicating that even the basal level of kinase activity is sufficient to reduce Na+ channel activity significantly. Na+ currents in membrane patches from kinase-deficient cells were reduced up to 80% by phosphorylation by cAMP-dependent protein kinase. These effects could be blocked by a specific peptide inhibitor of cAMP-dependent protein kinase and reversed by phosphoprotein phosphatases. Convergent modulation of brain Na+ channels by neurotransmitters acting through the cAMP and protein kinase C signaling pathways may result in associative regulation of electrical activity by different synaptic inputs.

Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II.

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor-trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.

Muscarinic modulation of sodium current by activation of protein kinase C in rat hippocampal neurons.

Phosphorylation of brain Na+ channels by protein kinase C (PKC) decreases peak Na+ current and slows macroscopic inactivation, but receptor-activated modulation of Na+ currents via the PKC pathway has not been demonstrated. We have examined modulation of Na+ channels by activation of muscarinic receptors in acutely-isolated hippocampal neurons using whole-cell voltage-clamp recording. Application of the muscarinic agonist carbachol reduced peak Na+ current and slowed macroscopic inactivation at all potentials, without changing the voltage-dependent properties of the channel. These effects were mediated by PKC, since they were eliminated when the specific PKC inhibitor (PKCI19-36) was included in the pipette solution and mimicked by the extracellular application of the PKC activator, OAG. Thus, activation of endogenous muscarinic receptors on hippocampal neurons strongly modulates Na+ channel activity by activation of PKC. Cholinergic input from basal forebrain neurons may have this effect in the hippocampus in vivo.

Primary structure and function of an A kinase anchoring protein associated with calcium channels.

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.

A sodium channel signaling complex: modulation by associated receptor protein tyrosine phosphatase beta.

Voltage-gated sodium channels in brain neurons were found to associate with receptor protein tyrosine phosphatase beta (RPTPbeta) and its catalytically inactive, secreted isoform phosphacan, and this interaction was regulated during development. Both the extracellular domain and the intracellular catalytic domain of RPTPbeta interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of RPTPbeta. Dephosphorylation slowed sodium channel inactivation, positively shifted its voltage dependence, and increased whole-cell sodium current. Our results define a sodium channel signaling complex containing RPTPbeta, which acts to regulate sodium channel modulation by tyrosine phosphorylation.

Dopaminergic modulation of voltage-gated Na+ current in rat hippocampal neurons requires anchoring of cAMP-dependent protein kinase.

Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in acutely isolated hippocampal neurons via a modulatory mechanism involving phosphorylation of the Na(+) channel alpha subunit by cAMP-dependent protein kinase (PKA). Peak Na(+) current is reduced 20-50% in the presence of the D1 agonist SKF 81297 or the PKA activator Sp-5,6-dichloro-l-beta-d-ribofuranosyl benzimidazole-3',5'-cyclic monophosphorothionate (cBIMPS). Co-immunoprecipitation experiments show that Na(+) channels are associated with PKA and A-kinase-anchoring protein 15 (AKAP-15), and immunocytochemical labeling reveals their co-localization in the cell bodies and proximal dendrites of hippocampal pyramidal neurons. Anchoring of PKA near the channel by an AKAP, which binds the RII alpha regulatory subunit, is necessary for Na(+) channel modulation in acutely dissociated hippocampal pyramidal neurons. Intracellular dialysis with the anchoring inhibitor peptides Ht31 from a human thyroid AKAP and AP2 from AKAP-15 eliminated the modulation of the Na(+) channel by the D1-agonist SKF 81297 and the PKA activator cBIMPS. In contrast, dialysis with the inactive proline-substituted control peptides Ht31-P and AP2-P had little effect on the D1 and PKA modulation. Therefore, we conclude that modulation of the Na(+) channel by activation of D1-like DA receptors requires targeted localization of PKA near the channel to achieve phosphorylation of the alpha subunit and to modify the functional properties of the channel.

Contractile activation in scorpion striated muscle fibers. Dependence on voltage and external calcium.

Excitation-contraction coupling was characterized in scorpion striated muscle fibers using standard microelectrode techniques as employed in studies on vertebrate skeletal muscle. The action potential of scorpion muscle consists of two phases of regenerative activity. A relatively fast, overshooting initial spike is followed by a prolonged after-discharge of smaller, repetitive spikes. This after-discharge is accompanied by a twitch that relaxes promptly upon repolarization. Twitches fail in Na-free, tetrodotoxin (TTX)-containing, or Ca-free media. However, caffeine causes contractures in muscles paralyzed by Na- and Ca-free solutions. Experiments on muscle fibers voltage-clamped at a point with two microelectrodes in Na-free or TTX-containing media indicate that: (a) the strength-duration relation for threshold contractions has a shape similar to that in frog muscle, but mean values are displaced approximately 20 mV in the positive direction; (b) tetracaine exerts a parallel effect on strength-duration curves from scorpion and frog; (c) contractile activation in scorpion is abolished in Ca-free media; and (d) the contractile threshold is highly correlated with the occurrence of inward Ca current for pulses of all durations. Thus, the voltage dependence of contractile activation in scorpion and frog muscle is similar. However, the preparations differ in their dependence on extracellular Ca for contraction. These results are discussed in relation to possible mechanisms coupling tubular depolarization to Ca release from the sarcoplasmic reticulum in vertebrate and invertebrate skeletal muscle.

Block of outward current in cardiac Purkinje fibers by injection of quaternary ammonium ions.

We have studied the effects of iontophoretic injection of the quaternary ammonium compounds tetraethylammonium (TEA) and tetrabutylammonium (TBA) in cardiac purkinje fibers. We find that TBA(+) is a more effective blocker than TEA(+), but injection of either compound reduces the time-dependent outward plateau currents, transient outward current (I(to)), and the delayed rectifier (I(x)). Our findings provide evidence that these outward cardiac currents are carried by channels that in some respects are pharmacologically similar to squid axon potassium channels. We demonstrate that this procedure is a new tool that can be useful in the analysis of membrane currents in the heart.

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin.

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.