RESUMO
Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation, but the underlying ionic mechanism for this association remains unclear. We recently reported that expression of the small-conductance calcium-activated potassium channel 2 (SK2, encoded by KCCN2) in atria from diabetic mice is significantly down-regulated, resulting in reduced SK currents in atrial myocytes from these mice. We also reported that the level of SK2 mRNA expression is not reduced in DM atria but that the ubiquitin-proteasome system (UPS), a major mechanism of intracellular protein degradation, is activated in vascular smooth muscle cells in DM. This suggests a possible role of the UPS in reduced SK currents. To test this possibility, we examined the role of the UPS in atrial SK2 down-regulation in DM. We found that a muscle-specific E3 ligase, F-box protein 32 (FBXO-32, also called atrogin-1), was significantly up-regulated in diabetic mouse atria. Enhanced FBXO-32 expression in atrial cells significantly reduced SK2 protein expression, and siRNA-mediated FBXO-32 knockdown increased SK2 protein expression. Furthermore, co-transfection of SK2 with FBXO-32 complementary DNA in HEK293 cells significantly reduced SK2 expression, whereas co-transfection with atrogin-1ΔF complementary DNA (a nonfunctional FBXO-32 variant in which the F-box domain is deleted) did not have any effects on SK2. These results indicate that FBXO-32 contributes to SK2 down-regulation and that the F-box domain is essential for FBXO-32 function. In conclusion, DM-induced SK2 channel down-regulation appears to be due to an FBXO-32-dependent increase in UPS-mediated SK2 protein degradation.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Regulação para Baixo , Proteínas Musculares/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Camundongos , Proteínas Musculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Estreptozocina , Células Tumorais Cultivadas , Ubiquitina/metabolismoRESUMO
The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.
RESUMO
Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from MECP2 gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of Mecp2 mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Neurônios/fisiologia , RNA/genética , Adenosina Desaminase/genética , Animais , Células Cultivadas , Metilação de DNA/genética , Modelos Animais de Doenças , Humanos , Camundongos , Mutação/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Síndrome de Rett/genéticaRESUMO
Exome sequencing from a patient with neurological and developmental symptoms revealed two mutations in separate genes. One was a homozygous transition mutation that results in an in-frame, premature translational stop codon in the ZNF135 gene predicted to encode a transcriptional repressor. Another mutation was heterozygous, a single nucleotide duplication in the KCNN2 gene that encodes a Ca-activated K channel, SK2, and leads to a translational frame shift and a premature stop codon. Heterologous expression studies, brain slice recordings, and coordination tests from a transgenic mouse line carrying the SK2 mutation suggest that it does not contribute to the patient's symptoms. ZNF135 is expressed in human brain and it is likely that the homozygous mutation underlies the human phenotype.
Assuntos
Mutação , Doenças do Sistema Nervoso/genética , Proteínas Repressoras/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Adulto , Animais , Sistemas CRISPR-Cas , Estudos de Coortes , Feminino , Edição de Genes , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Mutagênese Sítio-Dirigida , Doenças do Sistema Nervoso/fisiopatologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Técnicas de Cultura de TecidosRESUMO
Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca(2+)-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study, a novel endothelium-specific SK3 knockout (SK3(-/-)) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca(2+) dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time quantitative PCR and Western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3(-/-) small mesenteric arteries. Immunostaining results from SK3(-/-) vessels confirmed the absence of SK3 and further showed altered distribution of transient receptor potential channels, type 4 (TRPV4). Electrophysiological recordings showed a lack of SK3 channel activity, while TRPV4-IK1 channel coupling remained intact in SK3(-/-) endothelial cells. Moreover, Ca(2+) imaging studies in SK3(-/-) endothelium showed increased Ca(2+) transients with reduced amplitude and duration under basal conditions. Importantly, SK3(-/-) endothelium lacked a distinct type of Ca(2+) dynamic that is sensitive to TRPV4 activation. Blood pressure measurements showed that the SK3(-/-) mice were hypertensive, and the blood pressure increase was further enhanced during the 12-h dark cycle when animals are most active. Taken together, our results reveal a previously unappreciated SK3 signaling microdomain that modulates endothelial Ca(2+) dynamics, vascular tone, and blood pressure.
Assuntos
Pressão Sanguínea , Sinalização do Cálcio , Cálcio/metabolismo , Células Endoteliais/metabolismo , Hipertensão/metabolismo , Microdomínios da Membrana/metabolismo , Artérias Mesentéricas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Vasodilatação , Ciclos de Atividade , Animais , Predisposição Genética para Doença , Hipertensão/genética , Hipertensão/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Cátion TRPV/metabolismo , Fatores de TempoRESUMO
The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2(-/-) mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity-by altering the Purkinje cell output-may be crucial to cerebellar information storage and learning.
Assuntos
Células de Purkinje/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Apamina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Baixa/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genéticaRESUMO
Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
Assuntos
Região CA1 Hipocampal/fisiologia , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Células Piramidais/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Linhagem Celular , Guanilato Quinases/isolamento & purificação , Humanos , Imunoprecipitação , Proteínas de Membrana/isolamento & purificação , Camundongos , Ligação Proteica , Canais de Potássio Ativados por Cálcio de Condutância Baixa/isolamento & purificaçãoRESUMO
In pyramidal neurons such as hippocampal area CA1 and basolateral amygdala, a slow afterhyperpolarization (sAHP) follows a burst of action potentials, which is a powerful regulator of neuronal excitability. The sAHP amplitude increases with aging and may underlie age related memory decline. The sAHP is due to a Ca(2+)-dependent, voltage-independent K(+) conductance, the molecular identity of which has remained elusive until a recent report suggested the Ca(2+)-activated K(+) channel, IK1 (KCNN4) as the sAHP channel in CA1 pyramidal neurons. The signature pharmacology of IK1, blockade by TRAM-34, was reported for the sAHP and underlying current. We have examined the sAHP and find no evidence that TRAM-34 affects either the current underling the sAHP or excitability of CA1 or basolateral amygdala pyramidal neurons. In addition, CA1 pyramidal neurons from IK1 null mice exhibit a characteristic sAHP current. Our results indicate that IK1 channels do not mediate the sAHP in pyramidal neurons.
Assuntos
Potenciais de Ação , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Piramidais/fisiologia , Animais , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pirazóis/metabolismo , Ratos WistarRESUMO
Mono-ADP-ribosylation (MARylation) of mammalian proteins was first described as a post-translational modification catalyzed by bacterial toxins. It is now known that endogenous MARylation occurs in mammalian cells and is catalyzed by 11 members of the poly-ADP-ribose polymerase (PARP) family of proteins (17 in humans). The physiological roles of these PARPs remain largely unknown. Here we demonstrate that PARP6, a neuronally enriched PARP that catalyzes MARylation, regulates hippocampal dendrite morphogenesis, a process that is critical for proper neural circuit formation during development. Knockdown of PARP6 significantly decreased dendritic complexity in embryonic rat hippocampal neurons in culture and in vivo. Expression of wild-type PARP6 increased dendritic complexity; conversely, expression of a catalytically inactive PARP6 mutant, or a cysteine-rich domain deletion mutant that has significantly reduced catalytic activity, decreased dendritic complexity. The identification of PARP6 as a regulator of dendrite morphogenesis supports a role for MARylation in neurons during development.
Assuntos
Dendritos/enzimologia , Hipocampo/enzimologia , Poli(ADP-Ribose) Polimerases/fisiologia , ADP Ribose Transferases , Animais , Células Cultivadas , Expressão Gênica , Hipocampo/embriologia , Camundongos Endogâmicos C57BL , Morfogênese , Neurogênese , Cultura Primária de Células , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ratos Sprague-DawleyRESUMO
Calcium ions are Nature's most widely used signaling mechanism, mediating communication between pathways at virtually every physiological level. Ion channels are no exception, as the activities of a wide range of ion channels are intricately shaped by fluctuations in intracellular Ca(2+) levels. Mirroring the importance and the breadth of Ca(2+) signaling, free Ca(2+) levels are tightly controlled, and a myriad of Ca(2+) binding proteins transduce Ca(2+) signals, each with its own nuance, comprising a constantly changing symphony of metabolic activity. The founding member of Ca(2+) binding proteins is calmodulin (CaM), a small, acidic, modular protein endowed with gymnastic-like flexibility and E-F hand motifs that chelate Ca(2+) ions. In this review, I will trace the history that led to the realization that CaM serves as the Ca(2+)-gating cue for SK channels, the experiments that revealed that CaM is an intrinsic subunit of SK channels, and itself a target of regulation.
Assuntos
Calmodulina/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Apamina/metabolismo , Cálcio/metabolismo , Humanos , Potássio/metabolismoRESUMO
SK2- and KV4.2-containing K+ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca2+ source that provides Ca2+-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca2+ entry through NMDARs, whereas KV4.2-containing channel availability is increased by Ca2+ entry through SNX-482 (SNX) sensitive CaV2.3 R-type Ca2+ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca2+ entry through R-type Ca2+ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca2+ channels and KV4.2-containing K+ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca2+ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from CaV2.3 null mice. The results show that in the absence of CaV2.3 channels, apamin application still boosted EPSPs. The boosting effect of CaV2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from CaV2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and KV4.2-containing channels to CaV2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.
Assuntos
Apamina/farmacologia , Canais de Cálcio Tipo R/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Células Piramidais/efeitos dos fármacos , Potenciais Sinápticos/efeitos dos fármacos , Animais , Região CA1 Hipocampal/citologia , Cálcio/metabolismo , Canais de Cálcio Tipo R/deficiência , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Potenciais Evocados/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Venenos de Aranha/farmacologia , Potenciais Sinápticos/genética , Potenciais Sinápticos/fisiologiaRESUMO
Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca(2+) wave-dependent activation of small-conductance Ca(2+)-activated K(+) (SK) channels. Here, we show that mGlu5 receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu5 receptor stimulation activated apamin-sensitive SK2-mediated K(+) currents. In addition, coexpression of mGlu5 receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu5 receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu5 receptors and SK2 channels that reflects their molecular coassembly.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Apamina/farmacologia , Cálcio/metabolismo , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/ultraestrutura , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , RatosRESUMO
Small conductance Ca(2+)-activated K(+) (SK) channels and voltage-gated A-type Kv4 channels shape dendritic excitatory postsynaptic potentials (EPSPs) in hippocampal CA1 pyramidal neurons. Synaptically evoked Ca(2+) influx through N-methyl-D-aspartate receptors (NMDARs) activates spine SK channels, reducing EPSPs and the associated spine head Ca(2+) transient. However, results using glutamate uncaging implicated Ca(2+) influx through SNX-482-sensitive (SNX-sensitive) Cav2.3 (R-type) Ca(2+) channels as the Ca(2+) source for SK channel activation. The present findings show that, using Schaffer collateral stimulation, the effects of SNX and apamin are not mutually exclusive and SNX increases EPSPs independent of SK channel activity. Dialysis with 1,2-bis(o-aminophenoxy)ethane-N'N'N'-tetraacetic acid (BAPTA), application of 4-Aminopyridine (4-AP), expression of a Kv4.2 dominant negative subunit, and dialysis with a KChIPs antibody occluded the SNX-induced increase of EPSPs. The results suggest two distinct Ca(2+) signaling pathways within dendritic spines that link Ca(2+) influx through NMDARs to SK channels and Ca(2+) influx through R-type Ca(2+) channels to Kv4.2-containing channels.
Assuntos
Região CA1 Hipocampal/citologia , Cálcio/metabolismo , Espinhas Dendríticas/fisiologia , Neurônios/citologia , Canais de Potássio Shal/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , 4-Aminopiridina/farmacologia , Animais , Animais Recém-Nascidos , Apamina/farmacologia , Espinhas Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estimulação Elétrica , Eletroporação , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas In Vitro , Camundongos , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Canais de Potássio Shal/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Venenos de Aranha , Fatores de TempoRESUMO
Small-conductance, Ca(2+) -activated K(+) (SK) channels are expressed in the hippocampus where they regulate synaptic responses, plasticity, and learning and memory. To investigate the expression of SK3 (KCNN3) subunits, we determined the developmental profile and subcellular distribution of SK3 in the developing mouse hippocampus using western blots, immunohistochemistry and high-resolution immunoelectron microscopy. The results showed that SK3 expression increased during postnatal development, and that the localization of SK3 changed from being mainly associated with the endoplasmic reticulum and intracellular sites during the first postnatal week to being progressively concentrated in dendritic spines during later stages. In the adult, SK3 was localized mainly in postsynaptic compartments, both at extrasynaptic sites and along the postsynaptic density of excitatory synapses. Double labelling showed that SK3 co-localized with SK2 (KCNN2) and with N-methyl-D-aspartate receptors. Finally, quantitative analysis of SK3 density revealed two subcellular distribution patterns in different hippocampal layers, with SK3 being unevenly distributed in CA1 region of the hippocampus pyramidal cells and homogeneously distributed in dentate gyrus granule cells. Our results revealed a complex cell surface distribution of SK3-containing channels and a distinct developmental program that may influence different hippocampal functions.
Assuntos
Região CA1 Hipocampal/metabolismo , Densidade Pós-Sináptica/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Região CA1 Hipocampal/crescimento & desenvolvimento , Dendritos/metabolismo , Retículo Endoplasmático/metabolismo , Camundongos , Especificidade de Órgãos , Transporte Proteico , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genéticaRESUMO
Small-conductance, Ca(2+)-activated K(+) (SK) channels regulate neuronal excitability in a variety of ways. To understand their roles in different neuronal subtypes it is important to determine their precise subcellular distribution. Here, we used biochemical, light microscopy immunohistochemical and immunoelectron microscopy techniques, combined with quantitative approaches, to reveal the expression and subcellular localization patterns of SK2 in the developing cerebellum. Using western blots, the SK2 protein showed a progressive increase during postnatal development. At the light microscopic level, SK2 immunoreactivity was very prominent in the developing Purkinje cells (PC), particularly in the molecular layer (ML). Electron microscopy revealed that throughout development SK2 was mostly detected at the extrasynaptic and perisynaptic plasma membrane of dendritic shafts and dendritic spines of PCs. However, there was some localization at axon terminals as well. Quantitative analyses and 3D reconstructions further revealed a progressive developmental change of SK2 on the surface of PCs from dendritic shafts to dendritic spines. Together, these results indicate that SK2 channels undergo dynamic spatial regulation during cerebellar development, and this process is associated with the formation and maturation of excitatory synaptic contacts to PCs.
RESUMO
Premature and long-term ovarian hormone loss following ovariectomy (OVX) is associated with cognitive impairment. This condition is prevented by estradiol (E2) therapy when initiated shortly following OVX but not after substantial delay. To determine whether these clinical findings are correlated with changes in synaptic functions, we used adult OVX rats to evaluate the consequences of short-term (7-10 d, OVXControl) and long-term (â¼5 months, OVXLT) ovarian hormone loss, as well as subsequent in vivo E2 treatment, on excitatory synaptic transmission at the hippocampal CA3-CA1 synapses important for learning and memory. The results show that ovarian hormone loss was associated with a marked decrease in synaptic strength. E2 treatment increased synaptic strength in OVXControl but not OVXLT rats, demonstrating a change in the efficacy for E2 5 months following OVX. E2 also had a more rapid effect: within minutes of bath application, E2 acutely increased synaptic strength in all groups except OVXLT rats that did not receive in vivo E2 treatment. E2's acute effect was mediated postsynaptically, and required Ca(2+) influx through the voltage-gated Ca(2+) channels. Despite E2's acute effect, synaptic strength of OVXLT rats remained significantly lower than that of OVXControl rats. Thus, changes in CA3-CA1 synaptic transmission associated with ovarian hormone loss cannot be fully reversed with delayed E2 treatment. Given that synaptic strength at CA3-CA1 synapses is related to the ability to learn hippocampus-dependent tasks, these findings provide additional insights for understanding cognitive impairment-associated long-term ovarian hormone loss and ineffectiveness for delayed E2 treatment to maintain cognitive functions.
Assuntos
Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/metabolismo , Estradiol/deficiência , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Western Blotting , Estradiol/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hormônios Esteroides Gonadais/deficiência , Hormônios Esteroides Gonadais/farmacologia , Ovariectomia , Técnicas de Patch-Clamp , Ratos , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
The development of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development. In the developing mammalian auditory system, spontaneous calcium action potentials are generated by inner hair cells (IHCs), which form the primary sensory synapse. It remains unknown whether this electrical activity is required for the functional maturation of the auditory system. We found that sensory-independent electrical activity controls synaptic maturation in IHCs. We used a mouse model in which the potassium channel SK2 is normally overexpressed, but can be modulated in vivo using doxycycline. SK2 overexpression affected the frequency and duration of spontaneous action potentials, which prevented the development of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their morphology or general cell development. By manipulating the in vivo expression of SK2 channels, we identified the "critical period" during which spiking activity influences IHC synaptic maturation. Here we provide direct evidence that IHC development depends upon a specific temporal pattern of calcium spikes before sound-driven neuronal activity.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Sinapses/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Doxiciclina/farmacologia , Células Ciliadas Auditivas Internas/citologia , Camundongos , Camundongos Transgênicos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Sinapses/genéticaRESUMO
Sleep spindles are synchronized 11-15 Hz electroencephalographic (EEG) oscillations predominant during nonrapid-eye-movement sleep (NREMS). Rhythmic bursting in the reticular thalamic nucleus (nRt), arising from interplay between Ca(v)3.3-type Ca(2+) channels and Ca(2+)-dependent small-conductance-type 2 (SK2) K(+) channels, underlies spindle generation. Correlative evidence indicates that spindles contribute to memory consolidation and protection against environmental noise in human NREMS. Here, we describe a molecular mechanism through which spindle power is selectively extended and we probed the actions of intensified spindling in the naturally sleeping mouse. Using electrophysiological recordings in acute brain slices from SK2 channel-overexpressing (SK2-OE) mice, we found that nRt bursting was potentiated and thalamic circuit oscillations were prolonged. Moreover, nRt cells showed greater resilience to transit from burst to tonic discharge in response to gradual depolarization, mimicking transitions out of NREMS. Compared with wild-type littermates, chronic EEG recordings of SK2-OE mice contained less fragmented NREMS, while the NREMS EEG power spectrum was conserved. Furthermore, EEG spindle activity was prolonged at NREMS exit. Finally, when exposed to white noise, SK2-OE mice needed stronger stimuli to arouse. Increased nRt bursting thus strengthens spindles and improves sleep quality through mechanisms independent of EEG slow waves (<4 Hz), suggesting SK2 signaling as a new potential therapeutic target for sleep disorders and for neuropsychiatric diseases accompanied by weakened sleep spindles.
Assuntos
Nível de Alerta/fisiologia , Fases do Sono/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Núcleos Talâmicos/fisiologia , Potenciais de Ação , Animais , Limiar Auditivo , Células Cultivadas/fisiologia , Eletroencefalografia , Feminino , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Polissonografia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/biossíntese , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Organismos Livres de Patógenos Específicos , Núcleos Talâmicos/citologia , Regulação para CimaRESUMO
The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Órgão Vomeronasal/fisiologia , Agressão/fisiologia , Animais , Comportamento Animal/fisiologia , Dendritos/fisiologia , Feminino , Imunofluorescência , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muco/metabolismo , Técnicas de Patch-Clamp , Potássio/farmacologia , Comportamento Sexual Animal/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Estimulação Química , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/fisiologia , Urina/fisiologiaRESUMO
Small-conductance Ca(2+)-activated K(+) channels (SK channels) modulate excitability and curtail excitatory postsynaptic potentials (EPSPs) in neuronal dendrites. Here, we demonstrate long-lasting plasticity of intrinsic excitability (IE) in dendrites that results from changes in the gain of this regulatory mechanism. Using dendritic patch-clamp recordings from rat cerebellar Purkinje cells, we find that somatic depolarization or parallel fiber (PF) burst stimulation induce long-term amplification of synaptic responses to climbing fiber (CF) or PF stimulation and enhance the amplitude of passively propagated sodium spikes. Dendritic plasticity is mimicked and occluded by the SK channel blocker apamin and is absent in Purkinje cells from SK2 null mice. Triple-patch recordings from two dendritic sites and the soma and confocal calcium imaging studies show that local stimulation limits dendritic plasticity to the activated compartment of the dendrite. This plasticity mechanism allows Purkinje cells to adjust the SK2-mediated control of dendritic excitability in an activity-dependent manner.