RESUMO
The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.
Assuntos
Células T Matadoras Naturais , Serotonina , Serotonina/metabolismo , Lipídeos , Antígenos CD1d/metabolismoRESUMO
Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.
Assuntos
Mioquimia , Animais , Camundongos , Drosophila melanogaster , Ataxia , Drosophila , Canal de Potássio Kv1.2RESUMO
BACKGROUND: Autoantibodies against the potassium voltage-gated channel subfamily A member 2 (KCNA2) have been described in a few cases of neuropsychiatric disorders, but their diagnostic and pathophysiological role is currently unknown, imposing challenges to medical practice. DESIGN / METHODS: We retrospectively collected comprehensive clinical and paraclinical data of 35 patients with KCNA2 IgG autoantibodies detected in cell-based and tissue-based assays. Patients' sera and cerebrospinal fluid (CSF) were used for characterization of the antigen, clinical-serological correlations, and determination of IgG subclasses. RESULTS: KCNA2 autoantibody-positive patients (n = 35, median age at disease onset of 65 years, range of 16-83 years, 74 % male) mostly presented with cognitive impairment and/or epileptic seizures but also ataxia, gait disorder and personality changes. Serum autoantibodies belonged to IgG3 and IgG1 subclasses and titers ranged from 1:32 to 1:10,000. KCNA2 IgG was found in the CSF of 8/21 (38 %) patients and in the serum of 4/96 (4.2 %) healthy blood donors. KCNA2 autoantibodies bound to characteristic anatomical areas in the cerebellum and hippocampus of mammalian brain and juxtaparanodal regions of peripheral nerves but reacted exclusively with intracellular epitopes. A subset of four KCNA2 autoantibody-positive patients responded markedly to immunotherapy alongside with conversion to seronegativity, in particular those presenting an autoimmune encephalitis phenotype and receiving early immunotherapy. An available brain biopsy showed strong immune cell invasion. KCNA2 autoantibodies occurred in less than 10 % in association with an underlying tumor. CONCLUSION: Our data suggest that KCNA2 autoimmunity is clinically heterogeneous. Future studies should determine whether KCNA2 autoantibodies are directly pathogenic or develop secondarily. Early immunotherapy should be considered, in particular if autoantibodies occur in CSF or if clinical or diagnostic findings suggest ongoing inflammation. Suspicious clinical phenotypes include autoimmune encephalitis, atypical dementia, new-onset epilepsy and unexplained epileptic seizures.
Assuntos
Doenças Autoimunes do Sistema Nervoso , Autoimunidade , Encefalite , Doença de Hashimoto , Animais , Humanos , Masculino , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Feminino , Estudos Retrospectivos , Autoanticorpos , Convulsões , Mamíferos , Canal de Potássio Kv1.2RESUMO
Autoimmune-mediated encephalitis syndromes are increasingly being recognized as important clinical entities. They need to be thought of as differential diagnosis in any patient presenting with fast-onset psychosis or psychiatric problems, memory deficits or other cognitive problems, including aphasias, as well as seizures or motor automatisms, but also rigidity, paresis, ataxia or dystonic / parkinsonian symptoms. Diagnosis including imaging and CSF search for antibodies needs to be fast, as progression of these inflammatory processes is often causing scarring of brain tissue, with hypergliosis and atrophy. As these symptoms show, the autoantibodies present in these cases appear to act within the CNS. Several of such antibodies have by now been identified such as IgG directed against NMDA-receptors, AMPA receptors, GABAA and GABAB receptors, and voltage gated potassium channels and proteins of the potassium channel complex (i.e. LGI1 and CASPR2). These are neuropil / surface antigens where antibody interaction can well be envisaged to cause dysfunction of the target protein, including internalization. Others, such as antibodies directed against GAD65 (an intracellular enzyme responsible for GABA-synthesis from glutamate), are discussed to constitute epiphenomena, but not causal agents in disease progression. This review will focus on the current knowledge of antibody interaction mechanisms, especially discussing cellular excitability changes and synaptic interactions in hippocampal and other brain networks. One challenge in this context is to find viable hypotheses for the emergence of both, hyperexcitability and seizures, and presumably reduced synaptic plasticity and underlying cognitive dysfunction.
Assuntos
Autoimunidade , Proteínas do Tecido Nervoso , Humanos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Autoanticorpos , Convulsões , Ácido gama-AminobutíricoRESUMO
In this paper, the influence of external terahertz electromagnetic fields with different frequencies of 4 THz, 10 THz, 15 THz, and 20 THz on the permeability of the Kv1.2 voltage-gated potassium ion channel on the nerve cell membrane was studied using the combined model of the "Constant Electric Field-Ion Imbalance" method by molecular dynamics. We found that although the applied terahertz electric field does not produce strong resonance with the -C=O groups of the conservative sequence T-V-G-Y-G amino acid residue of the selective filter (SF) of the channel, it would affect the stability of the electrostatic bond between potassium ions and the carbonyl group of T-V-G-Y-G of SF, and it would affect the stability of the hydrogen bond between water molecules and oxygen atoms of the hydroxyl group of the 374THR side chain at the SF entrance, changing the potential and occupied states of ions in the SF and the occurrence probability of the permeation mode of ions and resulting in the change in the permeability of the channel. Compared with no external electric field, when the external electric field with 15 THz frequency is applied, the lifetime of the hydrogen bond is reduced by 29%, the probability of the "soft knock on" mode is decreased by 46.9%, and the ion flux of the channel is activated by 67.7%. Our research results support the view that compared to "direct knock-on", "soft knock-on" is a slower permeation mode.
Assuntos
Campos Eletromagnéticos , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Simulação de Dinâmica Molecular , Íons/metabolismo , Permeabilidade , Potássio/metabolismo , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.5/metabolismoRESUMO
2-Aminoethoxydiphenyl borate (2-APB), a boron-containing compound, is a multitarget compound with potential as a drug precursor and exerts various effects in systems of the human body. Ion channels are among the reported targets of 2-APB. The effects of 2-APB on voltage-gated potassium channels (KV) have been reported, but the types of KV channels that 2-APB inhibits and the inhibitory mechanism remain unknown. In this paper, we discovered that 2-APB acted as an inhibitor of three representative human KV1 channels. 2-APB significantly blocked A-type Kv channel KV1.4 in a concentration-dependent manner, with an IC50 of 67.3 µM, while it inhibited the delayed outward rectifier channels KV1.2 and KV1.3, with IC50s of 310.4 µM and 454.9 µM, respectively. Further studies on KV1.4 showed that V549, T551, A553, and L554 at the cavity region and N-terminal played significant roles in 2-APB's effects on the KV1.4 channel. The results also indicated the importance of fast inactivation gating in determining the different effects of 2-APB on three channels. Interestingly, a current facilitation phenomenon by a short prepulse after 2-APB application was discovered for the first time. The docked modeling revealed that 2-APB could form hydrogen bonds with different sites in the cavity region of three channels, and the inhibition constants showed a similar trend to the experimental results. These findings revealed new molecular targets of 2-APB and demonstrated that 2-APB's effects on KV1 channels might be part of the reason for the diverse bioactivities of 2-APB in the human body and in animal models of human disease.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Humanos , Compostos de Boro/farmacologia , Canais IônicosRESUMO
The associative network of hippocampal CA3 is thought to contribute to rapid formation of contextual memory from one-trial learning, but the network mechanisms underlying decorrelation of neuronal ensembles in CA3 is largely unknown. Kv1.2 expressions in rodent CA3 pyramidal cells (CA3-PCs) are polarized to distal apical dendrites, and its downregulation specifically enhances dendritic responses to perforant pathway (PP) synaptic inputs. We found that haploinsufficiency of Kv1.2 (Kcna2+/-) in CA3-PCs, but not Kv1.1 (Kcna1+/-), lowers the threshold for long-term potentiation (LTP) at PP-CA3 synapses, and that the Kcna2+/- mice are normal in discrimination of distinct contexts but impaired in discrimination of similar but slightly distinct contexts. We further examined the neuronal ensembles in CA3 and dentate gyrus (DG), which represent the two similar contexts using in situ hybridization of immediate early genes, Homer1a and Arc. The size and overlap of CA3 ensembles activated by the first visit to the similar contexts were not different between wild type and Kcna2+/- mice, but these ensemble parameters diverged over training days between genotypes, suggesting that abnormal plastic changes at PP-CA3 synapses of Kcna2+/- mice is responsible for the impaired pattern separation. Unlike CA3, DG ensembles were not different between two genotype mice. The DG ensembles were already separated on the first day, and their overlap did not further evolve. Eventually, the Kcna2+/- mice exhibited larger CA3 ensemble size and overlap upon retrieval of two contexts, compared to wild type or Kcna1+/- mice. These results suggest that sparse LTP at PP-CA3 synapse probably supervised by mossy fiber inputs is essential for gradual decorrelation of CA3 ensembles.
Assuntos
Aprendizagem por Discriminação , Fibras Musgosas Hipocampais , Animais , Potenciação de Longa Duração/fisiologia , Camundongos , Fibras Musgosas Hipocampais/fisiologia , Via Perfurante , Células Piramidais/fisiologia , Sinapses/fisiologiaRESUMO
The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.
Assuntos
Conotoxinas , Gânglios Espinais , Potenciais da Membrana , Simulação de Dinâmica Molecular , Neurônios , Superfamília Shaker de Canais de Potássio , Conotoxinas/química , Conotoxinas/metabolismo , Gânglios Espinais/química , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Transporte de Íons , Neurônios/química , Neurônios/metabolismo , Ligação Proteica , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/metabolismoRESUMO
Voltage-gated potassium channels (Kv) are important regulators of neuronal excitability for its role of regulating resting membrane potential and repolarization. Recent studies show that Kv channels participate in neuropathic pain, but the detailed underlying mechanisms are far from being clear. In this study, we used siRNA, miR-137 agomir, and antagomir to regulate the expression of Kv1.2 in spinal cord and dorsal root ganglia (DRG) of naïve and chronic constriction injury (CCI) rats. Kv currents and neuron excitability in DRG neurons were examined by patch-clamp whole-cell recording to verify the change in Kv1.2 function. The results showed that Kv1.2 was down-regulated in DRG and spinal dorsal horn (SDH) by CCI. Knockdown of Kv1.2 by intrathecally injecting Kcna2 siRNA induced significant mechanical and thermal hypersensitivity in naïve rats. Concomitant with the down-regulation of Kv1.2 was an increase in the expression of the miR-137. The targeting and regulating of miR-137 on Kcna2 was verified by dual-luciferase reporter system and intrathecal injecting miR-137 agomir. Furthermore, rescuing the expression of Kv1.2 in CCI rats, achieved through inhibiting miR-137, restored the abnormal Kv currents and excitability in DRG neurons, and alleviated mechanical allodynia and thermal hyperalgesia. These results indicate that the miR-137-mediated Kv1.2 impairment is a crucial etiopathogenesis for the nerve injury-induced neuropathic pain and can be a novel potential therapeutic target for neuropathic pain management.
Assuntos
Canal de Potássio Kv1.2/metabolismo , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Animais , Epigênese Genética , Gânglios Espinais/metabolismo , Masculino , MicroRNAs/metabolismo , Neuralgia/etiologia , Neurônios/metabolismo , Traumatismos dos Nervos Periféricos/complicações , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesões , Corno Dorsal da Medula Espinal/metabolismoRESUMO
Activation of transient receptor potential vanilloid 4 (TRPV4) can increase hippocampal neuronal excitability. TRPV4 has been reported to be involved in the pathogenesis of epilepsy. Voltage-gated potassium channels (VGPCs) play an important role in regulating neuronal excitability and abnormal VGPCs expression or function is related to epilepsy. Here, we examined the effect of TRPV4 activation on the delayed rectifier potassium current (IK ) in hippocampal pyramidal neurons and on the Kv subunits expression in male mice. We also explored the role of TRPV4 in changes in Kv subunits expression in male mice following pilocarpine-induced status epilepticus (PISE). Application of TRPV4 agonists, GSK1016790A and 5,6-EET, markedly reduced IK in hippocampal pyramidal neurons and shifted the voltage-dependent inactivation curve to the hyperpolarizing direction. GSK1016790A- and 5,6-EET-induced inhibition of IK was blocked by TRPV4 specific antagonists, HC-067047 and RN1734. GSK1016790A-induced inhibition of IK was markedly attenuated by calcium/calmodulin-dependent kinase II (CaMKII) antagonist. Application of GSK1016790A for up to 1 hr did not change the hippocampal protein levels of Kv1.1, Kv1.2, or Kv2.1. Intracerebroventricular injection of GSK1016790A for 3 d reduced the hippocampal protein levels of Kv1.2 and Kv2.1, leaving that of Kv1.1 unchanged. Kv1.2 and Kv2.1 protein levels as well as IK reduced markedly in hippocampi on day 3 post PISE, which was significantly reversed by HC-067047. We conclude that activation of TRPV4 inhibits IK in hippocampal pyramidal neurons, possibly by activating CaMKII. TRPV4-induced decrease in Kv1.2 and Kv2.1 expression and IK may be involved in the pathological changes following PISE.
Assuntos
Canais de Potássio de Retificação Tardia/metabolismo , Células Piramidais/fisiologia , Estado Epiléptico/fisiopatologia , Canais de Cátion TRPV/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Canais de Potássio de Retificação Tardia/farmacologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfolinas/farmacologia , Pilocarpina , Células Piramidais/metabolismo , Pirróis/farmacologia , Estado Epiléptico/induzido quimicamente , Sulfonamidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
BACKGROUND: Nerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to neuropathic pain genesis. Eukaryotic initiation factor 4 gamma 2 (eIF4G2) is a general repressor of cap-dependent mRNA translation. Whether DRG eIF4G2 participates in nerve injury-induced alternations in gene expression and nociceptive hypersensitivity is unknown. METHODS: The expression and distribution of eIF4G2 mRNA and protein in mouse DRG after spinal nerve ligation (SNL) were assessed. Effects of eIF4G2 siRNA microinjected through a glass micropipette into the injured DRG on the SNL-induced DRG mu opioid receptor (MOR) and Kv1.2 downregulation and nociceptive hypersensitivity were examined. In addition, effects of DRG microinjection of adeno-associated virus 5-expressing eIF4G2 (AAV5-eIF4G2) on basal DRG MOR and Kv1.2 expression and nociceptive thresholds were analysed. RESULTS: eIF4G2 protein co-expressed with Kv1.2 and MOR in DRG neurones. Levels of eIF4G2 mRNA (1.7 [0.24] to 2.3 [0.14]-fold of sham, P<0.01) and protein (1.6 [0.14] to 2.5 [0.22]-fold of sham, P<0.01) in injured DRG were time-dependently increased on days 3-14 after SNL. Blocking increased eIF4G2 through microinjection of eIF4G2 siRNA into the injured DRG attenuated SNL-induced downregulation of DRG MOR and Kv1.2 and development and maintenance of nociceptive hypersensitivities. DRG microinjection of AAV5-eIF4G2 reduced DRG MOR and Kv1.2 expression and elicited hypersensitivities to mechanical, heat and cold stimuli in naïve mice. CONCLUSIONS: eIF4G2 contributes to neuropathic pain through participation in downregulation of Kv1.2 and MOR in injured DRG and is a potential target for treatment of this disorder.
Assuntos
Fator de Iniciação Eucariótico 4G/genética , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Canal de Potássio Kv1.2/genética , Neuralgia/genética , Receptores Opioides mu/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Camundongos , Neuralgia/metabolismo , Neurônios/metabolismo , Medição da DorRESUMO
Pathogenic variants in KCNA2, encoding for the voltage-gated potassium channel Kv1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of-function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)-valine(406)-proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype-phenotype correlation, variability, and predicted functional impact of KCNA2 variants.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genótipo , Canal de Potássio Kv1.2 , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso , Substituição de Aminoácidos , Feminino , Humanos , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/metabolismo , Masculino , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismoRESUMO
Expressional changes of pain-associated genes in primary sensory neurons of DRG are critical for neuropathic pain genesis. DNA methyltransferase (DNMT)-triggered DNA methylation silences gene expression. We show here that DNMT1, a canonical maintenance methyltransferase, acts as the de novo DNMT and is required for neuropathic pain genesis likely through repressing at least DRG Kcna2 gene expression in male mice. Peripheral nerve injury upregulated DNMT1 expression in the injured DRG through the transcription factor cAMP response element binding protein-triggered transcriptional activation of Dnmt1 gene. Blocking this upregulation prevented nerve injury-induced DNA methylation within the promoter and 5'-untranslated region of Kcna2 gene, rescued Kcna2 expression and total Kv current, attenuated hyperexcitability in the injured DRG neurons, and alleviated nerve injury-induced pain hypersensitivities. Given that Kcna2 is a key player in neuropathic pain, our findings suggest that DRG DNMT1 may be a potential target for neuropathic pain management.SIGNIFICANCE STATEMENT In the present study, we reported that DNMT1, a canonical DNA maintenance methyltransferase, is upregulated via the activation of the transcription factor CREB in the injured DRG after peripheral nerve injury. This upregulation was responsible for nerve injury-induced de novo DNA methylation within the promoter and 5'-untranslated region of the Kcna2 gene, reductions in Kcna2 expression and Kv current and increases in neuronal excitability in the injured DRG. Since pharmacological inhibition or genetic knockdown of DRG DNMT1 alleviated nerve injury-induced pain hypersensitivities, DRG DNMT1 contributes to neuropathic pain genesis partially through repression of DRG Kcna2 gene expression.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Repressão Epigenética/fisiologia , Canal de Potássio Kv1.2/metabolismo , Neuralgia/metabolismo , Neurônios Aferentes/metabolismo , Animais , Gânglios Espinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismos dos Nervos Periféricos/metabolismoRESUMO
Autosomal dominant lateral temporal epilepsy (ADLTE) is an inherited syndrome caused by mutations in the leucine-rich glioma inactivated 1 (LGI1) gene. It is known that glutamatergic transmission is altered in LGI1 mutant mice, and seizures can be reduced by restoring LGI1 function. Yet, the mechanism underlying ADLTE is unclear. Here, we propose that seizures in male LGI1-/- mice are due to nonsynaptic epileptiform activity in cortical neurons. We examined the intrinsic excitability of pyramidal neurons in the temporal cortex of male LGI1-/- mice and found that the voltage-gated K+ channel Kv1.2 was significantly downregulated. We also found that cytosolic phospholipase A2 (cPLA2)-cyclooxygenase 2 (Cox2) signaling was enhanced in LGI1-/- mice. Interestingly, Cox2 inhibition effectively restored the dysregulated Kv1.2 and reduced the intrinsic excitability of pyramidal neurons. Moreover, in vivo injection of celecoxib, an FDA-approved nonsteroidal anti-inflammatory drug, rescued the defective Kv1.2 (an â¼1.9-fold increase), thereby alleviating the seizure susceptibility and extending the life of LGI1-/- mice by 5 d. In summary, we conclude that LGI1 deficiency dysregulates cPLA2-Cox2 signaling to cause hyperexcitability of cortical pyramidal neurons, and celecoxib is a potential agent to manage human ADLTE.SIGNIFICANCE STATEMENT Haploinsufficiency of the leucine-rich glioma inactivated 1 (LGI1) gene is the major pathogenic basis for ADLTE, an inherited syndrome with no cure to date. Existing studies suggest that altered glutamatergic transmission in the hippocampus causes this disease, but the data are paradoxical. We demonstrate that the loss of LGI1 decreases Kv1.2 expression, enhances intrinsic excitability, and thereby causes epilepsy. Interestingly, for the first time, we show that an FDA-approved drug, celecoxib, rescues the Kv1.2 defect and alleviates seizure susceptibility in LGI1-/- mice, as well as improving their survival. Thus, we suggest that celecoxib is a promising drug for the treatment of ADLTE patients.
Assuntos
Anticonvulsivantes/uso terapêutico , Celecoxib/uso terapêutico , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Epilepsia do Lobo Temporal/tratamento farmacológico , Convulsões/tratamento farmacológico , Potenciais de Ação , Animais , Anticonvulsivantes/farmacologia , Celecoxib/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Epilepsia do Lobo Temporal/genética , Peptídeos e Proteínas de Sinalização Intracelular , Canal de Potássio Kv1.2/metabolismo , Masculino , Camundongos , Fosfolipases A2/metabolismo , Proteínas/genética , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Convulsões/genéticaRESUMO
The transmission of normal sensory and/or acute noxious information requires intact expression of pain-associated genes within the pain pathways of nervous system. Expressional changes of these genes after peripheral nerve injury are also critical for neuropathic pain induction and maintenance. Methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, regulates gene transcriptional activity. We report here that MBD1 in the primary sensory neurons of DRG is critical for the genesis of acute pain and neuropathic pain as DRG MBD1-deficient mice exhibit the reduced responses to acute mechanical, heat, cold, and capsaicin stimuli and the blunted nerve injury-induced pain hypersensitivities. Furthermore, DRG overexpression of MBD1 leads to spontaneous pain and evoked pain hypersensitivities in the WT mice and restores acute pain sensitivities in the MBD1-deficient mice. Mechanistically, MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 is likely a key player under the conditions of acute pain and neuropathic pain.SIGNIFICANCE STATEMENT In the present study, we revealed that the mice with deficiency of methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, in the DRG displayed the reduced responses to acute noxious stimuli and the blunted neuropathic pain. We also showed that DRG overexpression of MBD1 produced the hypersensitivities to noxious stimuli in the WT mice and rescued acute pain sensitivities in the MBD1-deficient mice. We have also provided the evidence that MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 may participate in the genesis of acute pain and neuropathic pain likely through regulating DNMT3a-controlled Oprm1 and Kcna2 gene expression in the DRG neurons.
Assuntos
Dor Aguda/metabolismo , Proteínas de Ligação a DNA/biossíntese , Epigênese Genética/fisiologia , Canal de Potássio Kv1.2/biossíntese , Neuralgia/metabolismo , Receptores Opioides mu/biossíntese , Dor Aguda/genética , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Gânglios Espinais/química , Gânglios Espinais/metabolismo , Inativação Gênica/fisiologia , Canal de Potássio Kv1.2/antagonistas & inibidores , Canal de Potássio Kv1.2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/genética , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/genética , Células Receptoras Sensoriais/química , Células Receptoras Sensoriais/metabolismoRESUMO
Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations.
Assuntos
Ativação do Canal Iônico , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.2/metabolismo , Modelos Biológicos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Kv 1.2 are voltage-dependent potassium channels of great biological importance. Despite the existence of many reports considering structure - function relations of the Kv 1.2 channel's quantitative domains, some details of the voltage gating remain ambiguous, or even unknown. One of the examples is the range of the S4-S6 domains motions involved in channel activation and gating. Another important question is to what extent the channel geometry influences the observable channel conductance at different voltages, and what mechanism stands behind. Does the narrowing of the pore reduce the conductance by ohmic resistance growth? The answer is surprisingly negative. But it can be explained in an alternative way by considering the fluctuations. To address these problems, we formulate geometric models that mimic the generic features of voltage sensor movement and trigger the movement of the other domains involved in gating. We carry out a complete simulation of S4-S6 domains translations and tilts. The obtained pore profiles allow to estimate the (ohmic) conductance dependency on the voltage. From a family of analysed models, we choose the one most concurring with the experimental data. The results allow to suggest the most probable scenario of S4-S6 domains movement during channel activation by membrane depolarization.
Assuntos
Canal de Potássio Kv1.2/metabolismo , Animais , Humanos , Canal de Potássio Kv1.2/química , Potenciais da Membrana/fisiologia , Modelos Moleculares , Domínios ProteicosRESUMO
The voltage-gated potassium channels Kv1.1 and Kv1.2 that cluster at juxtaparanodal (JXP) regions are essential in the regulation of nerve excitability and play a critical role in axonal conduction. When demyelination occurs, Kv1.1/Kv1.2 activity increases, suppressing the membrane potential nearly to the equilibrium potential of K+, which results in an axonal conduction blockade. The recovery of K+-dependent communication signals and proper clustering of Kv1.1/Kv1.2 channels at JXP regions may directly reflect nerve regeneration following peripheral nerve injury. However, little is known about potassium channel expression and its relationship with the dynamic potassium ion distribution at the node of Ranvier during the regenerative process of peripheral nerve injury (PNI). In the present study, end-to-end neurorrhaphy (EEN) was performed using an in vivo model of PNI. The distribution of K+ at regenerating axons following EEN was detected by time-of-flight secondary-ion mass spectrometry. The specific localization and expression of Kv1.1/Kv1.2 channels were examined by confocal microscopy and western blotting. Our data showed that the re-establishment of K+ distribution and intensity was correlated with the functional recovery of compound muscle action potential morphology in EEN rats. Furthermore, the re-clustering of Kv1.1/1.2 channels 1 and 3 months after EEN at the nodal region of the regenerating nerve corresponded to changes in the K+ distribution. This study provided direct evidence of K+ distribution in regenerating axons for the first time. We proposed that the Kv1.1/Kv1.2 channels re-clustered at the JXP regions of regenerating axons are essential for modulating the proper patterns of K+ distribution in axons for maintaining membrane potential stability after EEN.
Assuntos
Axônios/metabolismo , Canal de Potássio Kv1.1/metabolismo , Canal de Potássio Kv1.2/metabolismo , Terminações Nervosas/metabolismo , Procedimentos Neurocirúrgicos , Potássio/metabolismo , Animais , Axônios/patologia , Íons/metabolismo , Masculino , Terminações Nervosas/patologia , Regeneração Nervosa , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/cirurgia , Ratos , Ratos Wistar , Espectrometria de Massa de Íon Secundário , Fatores de TempoRESUMO
Numerous experiments using ex vivo electrophysiology suggest that mammalian learning and memory involves regulation of voltage-gated ion channels in terms of changes in function. Yet, little is known about learning-related regulation of voltage-gated ion channels in terms of changes in expression. In two experiments, we examined changes in cell surface expression of the voltage-gated potassium channel alpha-subunit Kv1.2 in a discrete region of cerebellar cortex after eyeblink conditioning (EBC), a well-studied form of cerebellar-dependent learning. Kv1.2 in cerebellar cortex is expressed almost entirely in basket cells, primarily in the axon terminal pinceaux (PCX) region, and Purkinje cells, primarily in dendrites. Cell surface expression of Kv1.2 was measured using both multiphoton microscopy, which allowed measurement confined to the PCX region, and biotinylation/western blot, which measured total cell surface expression. In the first experiment, rats underwent three sessions of EBC, explicitly unpaired stimulus exposure, or context-only exposure and the results revealed a decrease in Kv1.2 cell surface expression in the unpaired group as measured with microscopy but no change as measured with western blot. In the second experiment, the same three training groups underwent only one half of a session of training, and the results revealed an increase in Kv1.2 cell surface expression in the unpaired group as measured with western blot but no change as measured with microscopy. In addition, rats in the EBC group that did not express conditioned responses (CRs) exhibited the same increase in Kv1.2 cell surface expression as the unpaired group. The overall pattern of results suggests that cell surface expression of Kv1.2 is changed with exposure to EBC stimuli in the absence, or prior to the emergence, of CRs.
Assuntos
Piscadela/fisiologia , Córtex Cerebelar/metabolismo , Condicionamento Clássico/fisiologia , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Canal de Potássio Kv1.2/metabolismo , Células de Purkinje/metabolismo , Animais , Comportamento Animal/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Change is intrinsic to nervous systems; change is required for learning and conditioning and occurs with disease progression, normal development, and aging. To better understand mammalian nervous systems and effectively treat nervous-system disorders, it is essential to track changes in relevant individual neurons. A critical challenge is to identify and characterize the specific cell types involved and the molecular-level changes that occur in each. Using an experimental strategy called constellation pharmacology, we demonstrate that we can define a specific somatosensory neuronal subclass, cold thermosensors, across different species and track changes in these neurons as a function of development. Cold thermosensors are uniformly responsive to menthol and innocuous cool temperature (17 °C), indicating that they express TRPM8 channels. A subset of cold thermosensors expressed α7 nicotinic acetylcholine receptors (nAChRs) but not other nAChR subtypes. Differences in temperature threshold of cold thermosensors correlated with functional expression of voltage-gated K channels Kv1.1/1.2: Relatively higher expression of KV1.1/1.2 channels resulted in a higher threshold response to cold temperature. Other signaling components varied during development and between species. In cold thermosensors of neonatal mice and rats, ATP receptors were functionally expressed, but the expression disappeared with development. This developmental change occurred earlier in low-threshold than high-threshold cold thermosensors. Most rat cold thermosensors expressed TRPA1 channels, whereas mouse cold thermosensors did not. The broad implications of this study are that it is now feasible to track changes in receptor and ion-channel expression in individual neuronal subclasses as a function of development, learning, disease, or aging.