Novel modulator of Na(V)1.1 and Na(V)1.2 Na channels in rat neuronal cells.
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ABSTRACT:
SUBMITTER: Mao H
PROVIDER: S-EPMC2894714 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The expression of voltage-gated sodium channels is regulated at multiple levels, and in this study we addressed the potential for alternative splicing of the Na(v)1.2, Na(v)1.3, Na(v)1.6 and Na(v)1.7 mRNAs. We isolated novel mRNA isoforms of Na(v)1.2 and Na(v)1.3 from adult mouse and rat dorsal root ganglia (DRG), Na(v)1.3 and Na(v)1.7 from adult mouse brain, and Na(v)1.7 from neonatal rat brain. These alternatively spliced isoforms introduce an additional exon (Na(v)1.2 exon 17A and topologically equivalent Na(v)1.7 exon 16A) or exon pair (Na(v)1.3 exons 17A and 17B) that contain an in-frame stop codon and result in predicted two-domain, truncated proteins. The mouse and rat orthologous exon sequences are highly conserved (94-100% identities), as are the paralogous Na(v)1.2 and Na(v)1.3 exons (93% identity in mouse) to which the Na(v)1.7 exon has only 60% identity. Previously, Na(v)1.3 mRNA has been shown to be upregulated in rat DRG following peripheral nerve injury, unlike the downregulation of all other sodium channel transcripts. Here we show that the expression of Na(v)1.3 mRNA containing exons 17A and 17B is unchanged in mouse following peripheral nerve injury (axotomy), whereas total Na(v)1.3 mRNA expression is upregulated by 33% (P=0.003), suggesting differential regulation of the alternatively spliced transcripts. The alternatively spliced rodent exon sequences are highly conserved in both the human and chicken genomes, with 77-89% and 72-76% identities to mouse, respectively. The widespread conservation of these sequences strongly suggests an additional level of regulation in the expression of these channels, that is also tissue-specific.
| S-EPMC2726981 | biostudies-literature
Project description:See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.
| S-EPMC5888929 | biostudies-literature
Project description:Voltage-gated sodium channels are important targets for modulation of electrical excitability by neurotransmitters and neurotrophins acting through protein phosphorylation. Fast inactivation of Na(V)1.2 channels is regulated via tyrosine phosphorylation by Fyn kinase and dephosphorylation by receptor phosphoprotein tyrosine phosphatase-beta, which are associated in a signaling complex. Here we have identified the amino acid residues on Na(V)1.2 channels that coordinate binding of Fyn kinase and mediate inhibition of sodium currents by enhancing fast inactivation. Fyn kinase binds to a Src homology 3 (SH3)-binding motif in the second half of the intracellular loop connecting domains I and II (L(I-II)) of Na(V)1.2, and mutation of that SH3-binding motif prevents Fyn binding and Fyn enhancement of fast inactivation of sodium currents. Analysis of tyrosine phosphorylation sites by mutagenesis and functional expression revealed a multisite regulatory mechanism. Y66 and Y1893, which are in consensus sequences appropriate for binding to the Fyn SH2 domain after phosphorylation, are both required for optimal binding and regulation by Fyn. Y730, which is located near the SH3-binding motif in L(I-II), and Y1497 and Y1498 in the inactivation gate in L(III-IV), are also required for optimal regulation. Phosphorylation of these sites likely promotes fast inactivation. Fast inactivation of the closely related Na(V)1.1 channels is not modulated by Fyn, and these channels do not contain an SH3-binding motif in L(I-II). Subtype-selective modulation by tyrosine phosphorylation/dephosphorylation provides a mechanism for differential regulation of sodium channels by neurotrophins and tyrosine phosphorylation in unmyelinated axons and dendrites, where Na(V)1.2 channels are expressed in brain neurons.
| S-EPMC6673227 | biostudies-literature
Project description:Na(V)1.1 is the primary voltage-gated Na(+) channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that Na(V)1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a(+/-)), which encodes the pore-forming ?-subunit of the Na(V)1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a(+/-) mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a(+/-) mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a(+/-) mice, and this communication is barely detectable in the homozygous gene KO (Scn1a(-/-)). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of Na(V)1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na(+) channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of Na(V)1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.
| S-EPMC3277539 | biostudies-literature
Project description:G protein gated inward rectifier K(+) (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four G?? subunits bind cooperatively to open GIRK2, and that intracellular Na(+) - which enters neurons during action potentials - further amplifies opening mostly by increasing G?? affinity. A Na(+) amplification function is characterized and used to estimate the concentration of G?? subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to G?? and Na(+), mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.
| S-EPMC4866826 | biostudies-literature
Project description:The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a ?-propeller and a classical ?/?-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions.
| S-EPMC4350108 | biostudies-literature
Project description:We have cloned and characterized mouse and human variants of MONaKA, a novel protein that interacts with and modulates the plasma membrane Na,K-ATPase. MONaKA was cloned based on its sequence homology to the Drosophila Slowpoke channel-binding protein dSlob, but mouse and human MONaKA do not bind to mammalian Slowpoke channels. At least two splice variants of MONaKA exist; the splicing is conserved perfectly between mouse and human, suggesting that it serves some important function. Both splice variants of MONaKA are expressed widely throughout the CNS and peripheral nervous system, with different splice variant expression ratios in neurons and glia. A yeast two-hybrid screen with MONaKA as bait revealed that it binds tightly to the beta1 and beta3 subunits of the Na,K-ATPase. The association between MONaKA and Na,K-ATPase beta subunits was confirmed further by coimmunoprecipitation from transfected cells, mouse brain, and cultured mouse astrocytes. A glutathione S-transferase-MONaKA fusion protein inhibits Na,K-ATPase activity from whole brain or cultured astrocytes. Furthermore, transfection of MONaKA inhibits 86Rb+ uptake via the Na,K-ATPase in intact cells. These results are consistent with the hypothesis that MONaKA modulates brain Na,K-ATPase and may thereby participate in the regulation of electrical excitability and synaptic transmission.
| S-EPMC6725465 | biostudies-literature
Project description:Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K(+) channels, K(v)1.1 and K(v)1.2 (voltage-gated K(+) channels) and TREK1 and TRAAK (K(2P) channels). IQ effectively reduced the currents mediated by both K(+) channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K(v)1.1 and K(v)1.2.Our results demonstrate that IQ blocks the voltage-gated K(+) channels to increase AP duration and K(2P) channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.
| S-EPMC3292985 | biostudies-literature
Project description:Scn2a encodes the voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability, resulting in epilepsy, whereas NaV1.2 deficiency impairs neuronal excitability, contributing to autism. However, this paradigm does not explain why ∼20%-30% of individuals with NaV1.2 deficiency still develop seizures. Here, we report the counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a NaV1.2-deficient mouse model, we show enhanced intrinsic excitability of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous and reversible by genetic restoration of Scn2a expression in adult mice. RNA sequencing reveals downregulation of multiple potassium channels, including KV1.1. Correspondingly, KV channel openers alleviate the hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.
| S-EPMC8382316 | 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