Sodium Channel SCN3A (NaV1.3) Regulation of Human Cerebral Cortical Folding and Oral Motor Development.
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ABSTRACT:
SUBMITTER: Smith RS
PROVIDER: S-EPMC6226006 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutations in the sodium channel genes SCN1A and SCN2A have been identified in monogenic childhood epilepsies, but SCN3A has not previously been investigated as a candidate gene for epilepsy. We screened a consecutive cohort of 18 children with cryptogenic partial epilepsy that was classified as pharmacoresistant because of nonresponse to carbamazepine or oxcarbazepine, antiepileptic drugs that bind sodium channels. The novel coding variant SCN3A-K354Q was identified in one patient and was not present in 295 neurological normal controls. Twelve novel SNPs were also detected. K354Q substitutes glutamine for an evolutionarily conserved lysine residue in the pore domain of SCN3A. Functional analysis of this mutation in the backbone of the closely related gene SCN5A demonstrated an increase in persistent current that is similar in magnitude to epileptogenic mutations of SCN1A and SCN2A. This observation of a potentially pathogenic mutation of SCN3A (Nav1.3) indicates that this gene should be further evaluated for its contribution to childhood epilepsy.
| S-EPMC2423278 | biostudies-literature
Project description:Background and purposeThe voltage-gated sodium channel Nav 1.7 is essential for adequate perception of painful stimuli. Mutations in the encoding gene, SCN9A, cause various pain syndromes in humans. The hNav 1.7/A1632E channel mutant causes symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD), and its main gating change is a strongly enhanced persistent current. On the basis of recently published 3D structures of voltage-gated sodium channels, we investigated how the inactivation particle binds to the channel, how this mechanism is altered by the hNav 1.7/A1632E mutation, and how dimerization modifies function of the pain-linked mutation.Experimental approachWe applied atomistic molecular simulations to demonstrate the effect of the mutation on channel fast inactivation. Native PAGE was used to demonstrate channel dimerization, and electrophysiological measurements in HEK cells and Xenopus laevis oocytes were used to analyze the links between functional channel dimerization and impairment of fast inactivation by the hNav 1.7/A1632E mutation.Key resultsEnhanced persistent current through hNav 1.7/A1632E channels was caused by impaired binding of the inactivation particle, which inhibits proper functioning of the recently proposed allosteric fast inactivation mechanism. hNav 1.7 channels form dimers and the disease-associated persistent current through hNav 1.7/A1632E channels depends on their functional dimerization status: Expression of the synthetic peptide difopein, a 14-3-3 inhibitor known to functionally uncouple dimers, decreased hNav 1.7/A1632E channel-induced persistent currents.Conclusion and implicationsFunctional uncoupling of mutant hNav 1.7/A1632E channel dimers restored their defective allosteric fast inactivation mechanism. Our findings support the concept of sodium channel dimerization and reveal its potential relevance for human pain syndromes.
| S-EPMC7484505 | biostudies-literature
Project description:Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
| S-EPMC8935509 | biostudies-literature
Project description:Loss-of-function variants in the gene SCN2A, which encodes the sodium channel NaV1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20%-30% of children with these variants also suffer from epilepsy, with altered neuronal activity originating in neocortex, a region where NaV1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking NaV1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic-clamp recordings revealed that NaV1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may, therefore, account for why SCN2A loss-of-function can paradoxically promote seizure.
| S-EPMC8719649 | biostudies-literature
Project description:µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7.
| S-EPMC9506549 | biostudies-literature
Project description:Homotetrameric bacterial voltage-gated sodium channels share major biophysical features with their more complex eukaryotic counterparts, including a slow-inactivation mechanism that reduces ion-conductance activity during prolonged depolarization through conformational changes in the pore. The bacterial sodium channel NaVAb activates at very negative membrane potentials and inactivates through a multiphase slow-inactivation mechanism. Early voltage-dependent inactivation during one depolarization is followed by late use-dependent inactivation during repetitive depolarization. Mutations that change the molecular volume of Thr206 in the pore-lining S6 segment can enhance or strongly block early voltage-dependent inactivation, suggesting that this residue serves as a molecular hub controlling the coupling of activation to inactivation. In contrast, truncation of the C-terminal tail enhances the early phase of inactivation yet completely blocks late use-dependent inactivation. Determination of the structure of a C-terminal tail truncation mutant and molecular modeling of conformational changes at Thr206 and the S6 activation gate led to a two-step model of these gating processes. First, bending of the S6 segment, local protein interactions dependent on the size of Thr206, and exchange of hydrogen-bonding partners at the level of Thr206 trigger pore opening followed by the early phase of voltage-dependent inactivation. Thereafter, conformational changes in the C-terminal tail lead to late use-dependent inactivation. These results have important implications for the sequence of conformational changes that lead to multiphase inactivation of NaVAb and other sodium channels.
| S-EPMC6363407 | biostudies-literature
Project description:Background and purposeThe development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7.Experimental approachEach VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings.Key resultsThe inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively.Conclusions and implicationsThe reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.
| S-EPMC5647187 | biostudies-literature
Project description:It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.
| S-EPMC10592450 | biostudies-literature
Project description:The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3- and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of NaV1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive NaV1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.
| S-EPMC7414216 | biostudies-literature
Project description:The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Nav1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Nav1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Nav1.5 variant Y87C and present first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.
| S-EPMC7515574 | biostudies-literature