RESUMO
Neurons in the suprachiasmatic nucleus (SCN) generate circadian changes in the rates of spontaneous action potential firing that regulate and synchronize daily rhythms in physiology and behavior. Considerable evidence suggests that daily rhythms in the repetitive firing rates (higher during the day than at night) of SCN neurons are mediated by changes in subthreshold potassium (K+) conductance(s). An alternative "bicycle" model for circadian regulation of membrane excitability in clock neurons, however, suggests that an increase in NALCN-encoded sodium (Na+) leak conductance underlies daytime increases in firing rates. The experiments reported here explored the role of Na+ leak currents in regulating daytime and nighttime repetitive firing rates in identified adult male and female mouse SCN neurons: vasoactive intestinal peptide-expressing (VIP+), neuromedin S-expressing (NMS+) and gastrin-releasing peptide-expressing (GRP+) cells. Whole-cell recordings from VIP+, NMS+, and GRP+ neurons in acute SCN slices revealed that Na+ leak current amplitudes/densities are similar during the day and at night, but have a larger impact on membrane potentials in daytime neurons. Additional experiments, using an in vivo conditional knockout approach, demonstrated that NALCN-encoded Na+ currents selectively regulate daytime repetitive firing rates of adult SCN neurons. Dynamic clamp-mediated manipulation revealed that the effects of NALCN-encoded Na+ currents on the repetitive firing rates of SCN neurons depend on K+ current-driven changes in input resistances. Together, these findings demonstrate that NALCN-encoded Na+ leak channels contribute to regulating daily rhythms in the excitability of SCN neurons by a mechanism that depends on K+ current-mediated rhythmic changes in intrinsic membrane properties.SIGNIFICANCE STATEMENT Elucidating the ionic mechanisms responsible for generating daily rhythms in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, is an important step toward understanding how the molecular clock controls circadian rhythms in physiology and behavior. While numerous studies have focused on identifying subthreshold K+ channel(s) that mediate day-night changes in the firing rates of SCN neurons, a role for Na+ leak currents has also been suggested. The results of the experiments presented here demonstrate that NALCN-encoded Na+ leak currents differentially modulate daily rhythms in the daytime/nighttime repetitive firing rates of SCN neurons as a consequence of rhythmic changes in subthreshold K+ currents.
Assuntos
Neurônios do Núcleo Supraquiasmático , Camundongos , Masculino , Feminino , Animais , Potenciais da Membrana/fisiologia , Potenciais de Ação/fisiologia , Ritmo Circadiano/fisiologia , Neurônios/fisiologia , Núcleo Supraquiasmático/fisiologia , Mamíferos , Canais Iônicos , Proteínas de MembranaRESUMO
RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.
Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/patologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Dobramento de Proteína , Tiorredoxinas/fisiologia , Fator 6 Ativador da Transcrição/biossíntese , Fator 6 Ativador da Transcrição/genética , Animais , Cardiomiopatia Hipertrófica/patologia , Células Cultivadas , Fibroblastos/patologia , Fibrose/metabolismo , Regulação da Expressão Gênica , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Humanos , Isoproterenol/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , NADPH Oxidase 4/biossíntese , NADPH Oxidase 4/genética , Células NIH 3T3 , Oxirredução , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genéticaRESUMO
Familial hypertrophic cardiomyopathy (HCM), linked to mutations in myosin, myosin-binding proteins and other sarcolemmal proteins, is associated with increased risk of life threatening ventricular arrhythmias, and a number of animal models have been developed to facilitate analysis of disease progression and mechanisms. In the experiments here, we use the αMHC403/+ mouse line in which one αMHC allele harbors a common HCM mutation (in ßMHC, Arg403 Gln). Here, we demonstrate marked prolongation of QT intervals in young adult (10-12week) male αMHC403/+ mice, well in advance of the onset of measurable left ventricular hypertrophy. Electrophysiological recordings from myocytes isolated from the interventricular septum of these animals revealed significantly (P<0.001) lower peak repolarizing voltage-gated K+ (Kv) current (IK,peak) amplitudes, compared with cells isolated from wild type (WT) littermate controls. Analysis of Kv current waveforms revealed that the amplitudes of the inactivating components of the total outward Kv current, Ito,f, Ito,s and IK,slow, were significantly lower in αMHC403/+, compared with WT, septum cells, whereas Iss amplitudes were similar. The amplitudes/densities of IK,peak and IK,slow were also lower in αMHC403/+, compared with WT, LV wall and LV apex myocytes, whereas Ito,f was attenuated in αMHC403/+ LV wall, but not LV apex, cells. These regional differences in the remodeling of repolarizing Kv currents in the αMHC403/+ mice would be expected to increase the dispersion of ventricular repolarization and be proarrhythmic. Quantitative RT-PCR analysis revealed reductions in the expression of transcripts encoding several K+ channel subunits in the interventricular septum, LV free wall and LV apex of (10-12week) αMHC403/+ mice, although this transcriptional remodeling was not correlated with the observed decreases in K+ current amplitudes.
Assuntos
Potenciais de Ação , Cardiomiopatia Hipertrófica Familiar/etiologia , Cardiomiopatia Hipertrófica Familiar/metabolismo , Mutação , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Miosinas Ventriculares/genética , Animais , Biópsia , Cardiomiopatia Hipertrófica Familiar/diagnóstico , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Expressão Gênica , Perfilação da Expressão Gênica , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genéticaRESUMO
Mutations in FGF14 , which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia type 27 (SCA27), a multisystem disorder associated with progressive deficits in motor coordination and cognitive function. Mice ( Fgf14 -/- ) lacking iFGF14 display similar phenotypes, and we have previously shown that the deficits in motor coordination reflect reduced excitability of cerebellar Purkinje neurons, owing to the loss of iFGF14-mediated regulation of the voltage-dependence of inactivation of the fast transient component of the voltage-gated Na + (Nav) current, I NaT . Here, we present the results of experiments designed to test the hypothesis that loss of iFGF14 also attenuates the intrinsic excitability of mature hippocampal and cortical pyramidal neurons. Current-clamp recordings from adult mouse hippocampal CA1 pyramidal neurons in acute in vitro slices, however, revealed that repetitive firing rates were higher in Fgf14 -/- , than in wild type (WT), cells. In addition, the waveforms of individual action potentials were altered in Fgf14 -/- hippocampal CA1 pyramidal neurons, and the loss of iFGF14 reduced the time delay between the initiation of axonal and somal action potentials. Voltage-clamp recordings revealed that the loss of iFGF14 altered the voltage-dependence of activation, but not inactivation, of I NaT in CA1 pyramidal neurons. Similar effects of the loss of iFGF14 on firing properties were evident in current-clamp recordings from layer 5 visual cortical pyramidal neurons. Additional experiments demonstrated that the loss of iFGF14 does not alter the distribution of anti-Nav1.6 or anti-ankyrin G immunofluorescence labeling intensity along the axon initial segments (AIS) of mature hippocampal CA1 or layer 5 visual cortical pyramidal neurons in situ . Taken together, the results demonstrate that, in contrast with results reported for neonatal (rat) hippocampal pyramidal neurons in dissociated cell culture, the loss of iFGF14 does not disrupt AIS architecture or Nav1.6 localization/distribution along the AIS of mature hippocampal (or cortical) pyramidal neurons in situ .
RESUMO
The fast transient outward K(+) current (Ito,f) underlies the early phase of myocardial action potential repolarization, contributing importantly to the coordinated propagation of activity in the heart and to the generation of normal cardiac rhythms. Native Ito,f channels reflect the tetrameric assembly of Kv4 pore-forming (α) subunits, and previous studies suggest roles for accessory and regulatory proteins in controlling the cell surface expression and the biophysical properties of Kv4-encoded Ito,f channels. Here, we demonstrate that the targeted deletion of the cytosolic accessory subunit, K(+) channel interacting protein 2 (KChIP2), results in the complete loss of the Kv4.2 protein, the α subunit critical for the generation of mouse ventricular Ito,f. Expression of the Kcnd2 (Kv4.2) transcript in KChIP2(-/-) ventricles, however, is unaffected. The loss of the Kv4.2 protein results in the elimination of Ito,f in KChIP2(-/-) ventricular myocytes. In parallel with the elimination of Ito,f, the slow transient outward K(+) current (Ito,s) is upregulated and voltage-gated Ca(2+) currents (ICa,L) are decreased. In addition, surface electrocardiograms and ventricular action potential waveforms in KChIP2(-/-) and wild-type mice are not significantly different, suggesting that the upregulation of Ito,s and the reduction in ICa,L compensate for the loss of Ito,f. Additional experiments revealed that Ito,f is not 'rescued' by adenovirus-mediated expression of KChIP2 in KChIP2(-/-) myocytes, although ICa,L densities are increased. Taken together, these results demonstrate that association with KChIP2 early in the biosynthetic pathway and KChIP2-mediated stabilization of Kv4 protein are critical determinants of native cardiac Ito,f channel expression.
Assuntos
Potenciais de Ação , Proteínas Interatuantes com Canais de Kv/metabolismo , Miócitos Cardíacos/fisiologia , Multimerização Proteica , Canais de Potássio Shal/metabolismo , Animais , Canais de Cálcio/metabolismo , Deleção de Genes , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Potássio Shal/genéticaRESUMO
Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K + ) channels regulate daily oscillations in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K + conductance(s) driving these daily rhythms in repetitive firing rates, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K + channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1 -/- ) or Kcnh3 (Kv12.2 -/- ) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1 -/- and Kv12.2 -/- , than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1 -/- , Kv12.2 -/- and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1 -/- and Kv12.2 -/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1 -/- , Kv12.2 -/- , Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.
RESUMO
Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I-IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11-iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.
Assuntos
Miócitos Cardíacos , Canais de Sódio , Camundongos , Ratos , Humanos , Animais , Canais de Sódio/metabolismo , Potenciais de Ação/fisiologia , Isoformas de Proteínas/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.
Assuntos
Neurônios do Núcleo Supraquiasmático , Animais , Camundongos , Mamíferos , Neurônios , Potássio , RNA Interferente Pequeno , Núcleo SupraquiasmáticoRESUMO
Native myocardial voltage-gated sodium (NaV) channels function in macromolecular complexes comprising a pore-forming (α) subunit and multiple accessory proteins. Here, we investigated the impact of accessory NaVß1 and NaVß3 subunits on the functional effects of 2 well-known class Ib antiarrhythmics, lidocaine and ranolazine, on the predominant NaV channel α subunit, NaV1.5, expressed in the mammalian heart. We showed that both drugs stabilized the activated conformation of the voltage sensor of domain-III (DIII-VSD) in NaV1.5. In the presence of NaVß1, the effect of lidocaine on the DIII-VSD was enhanced, whereas the effect of ranolazine was abolished. Mutating the main class Ib drug-binding site, F1760, affected but did not abolish the modulation of drug block by NaVß1/ß3. Recordings from adult mouse ventricular myocytes demonstrated that loss of Scn1b (NaVß1) differentially affected the potencies of lidocaine and ranolazine. In vivo experiments revealed distinct ECG responses to i.p. injection of ranolazine or lidocaine in WT and Scn1b-null animals, suggesting that NaVß1 modulated drug responses at the whole-heart level. In the human heart, we found that SCN1B transcript expression was 3 times higher in the atria than ventricles, differences that could, in combination with inherited or acquired cardiovascular disease, dramatically affect patient response to class Ib antiarrhythmic therapies.
Assuntos
Átrios do Coração , Lidocaína/farmacologia , Miócitos Cardíacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ranolazina/farmacologia , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Antiarrítmicos/farmacologia , Biomarcadores Farmacológicos/metabolismo , Eletrocardiografia/métodos , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Humanos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Tetrodotoxin (TTX) sensitive inward Ca2+ currents, ICa(TTX), have been identified in cardiac myocytes from several species, although it is unclear if ICa(TTX) is expressed in all cardiac cell types, and if ICa(TTX) reflects Ca2+ entry through the main, Nav1.5-encoded, cardiac Na+ (Nav) channels. To address these questions, recordings were obtained with 2 mm Ca2+ and 0 mm Na+ in the bath and 120 mm Cs+ in the pipettes from myocytes isolated from adult mouse interventricular septum (IVS), left ventricular (LV) endocardium, apex, and epicardium and from human LV endocardium and epicardium. On membrane depolarizations from a holding potential of -100 mV, ICa(TTX) was identified in mouse IVS and LV endocardial myocytes and in human LV endocardial myocytes, whereas only TTX-sensitive outward Cs+/K+ currents were observed in mouse LV apex and epicardial myocytes and human LV epicardial myocytes. The inward Ca2+, but not the outward Cs+/K+, currents were blocked by mm concentrations of MTSEA, a selective blocker of cardiac Nav1.5-encoded Na+ channels. In addition, in Nav1.5-expressing tsA-201 cells, ICa(TTX) was observed in 3 (of 20) cells, and TTX-sensitive outward Cs+/K+ currents were observed in the other (17) cells. The time- and voltage-dependent properties of the TTX-sensitive inward Ca2+ and outward Cs+/K+ currents recorded in Nav1.5-expressing tsA-201 were indistinguishable from native currents in mouse and human cardiac myocytes. Overall, the results presented here suggest marked regional, cell type-specific, differences in the relative ion selectivity, and likely the molecular architecture, of native SCN5A-/Scn5a- (Nav1.5-) encoded cardiac Na+ channels in mouse and human ventricles.
Assuntos
Cálcio/metabolismo , Césio/metabolismo , Condutividade Elétrica , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Potássio/metabolismo , Tetrodotoxina/farmacologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Sódio/metabolismoRESUMO
In cortical neurons, pore-forming alpha-subunits of the Kv4 subfamily underlie the fast transient outward K+ current (I(A)). Considerable evidence has accumulated demonstrating specific roles for I(A) channels in the generation of individual action potentials and in the regulation of repetitive firing. Although I(A) channels are thought to play a role in synaptic processing, little is known about the cell type- and synapse-specific distribution of these channels in cortical circuits. Here, we used immunolabeling with specific antibodies against Kv4.2 and Kv4.3, in combination with GABA immunogold staining, to determine the cellular, subcellular, and synaptic localization of Kv4 channels in the primary visual cortex of mice, in which subsets of pyramidal cells express yellow fluorescent protein. The results show that both Kv4.2 and Kv4.3 are concentrated in layer 1, the bottom of layer 2/3, and in layers 4 and 5/6. In all layers, clusters of Kv4.2 and Kv4.3 immunoreactivity are evident in the membranes of the somata, dendrites, and spines of pyramidal cells and GABAergic interneurons. Electron microscopic analyses revealed that Kv4.2 and Kv4.3 clusters in pyramidal cells and interneurons are excluded from putative excitatory synapses, whereas postsynaptic membranes at GABAergic synapses often contain Kv4.2 and Kv4.3. The presence of Kv4 channels at GABAergic synapses would be expected to weaken inhibition during dendritic depolarization by backpropagating action potentials. The extrasynaptic localization of Kv4 channels near excitatory synapses, in contrast, should stabilize synaptic excitation during dendritic depolarization. Thus, the synapse-specific distribution of Kv4 channels functions to optimize dendritic excitation and the association between presynaptic and postsynaptic activity.
Assuntos
Expressão Gênica/fisiologia , Neurônios/metabolismo , Canais de Potássio Shal/metabolismo , Sinapses/metabolismo , Córtex Visual/citologia , Animais , Proteínas de Bactérias/genética , Imuno-Histoquímica/métodos , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica/métodos , Neurônios/ultraestrutura , Canais de Potássio Shal/deficiência , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/metabolismoRESUMO
Rapidly activating and inactivating A-type K+ currents (IA) encoded by Kv4.2 and Kv4.3 pore-forming (α) subunits of the Kv4 subfamily are key regulators of neuronal excitability. Previous studies have suggested a role for Kv4.1 α-subunits in regulating the firing properties of mouse suprachiasmatic nucleus (SCN) neurons. To test this, we utilized an RNA-interference strategy to knockdown Kv4.1, acutely and selectively, in the SCN. Current-clamp recordings revealed that the in vivo knockdown of Kv4.1 significantly (p < 0.0001) increased mean ± SEM repetitive firing rates in SCN neurons during the day (6.4 ± 0.5 Hz) and at night (4.3 ± 0.6 Hz), compared with nontargeted shRNA-expressing SCN neurons (day: 3.1 ± 0.5 Hz; night: 1.6 ± 0.3 Hz). IA was also significantly (p < 0.05) reduced in Kv4.1-targeted shRNA-expressing SCN neurons (day: 80.3 ± 11.8 pA/pF; night: 55.3 ± 7.7 pA/pF), compared with nontargeted shRNA-expressing (day: 121.7 ± 10.2 pA/pF; night: 120.6 ± 16.5 pA/pF) SCN neurons. The magnitude of the effect of Kv4.1-targeted shRNA expression on firing rates and IA was larger at night. In addition, Kv4.1-targeted shRNA expression significantly (p < 0.001) increased mean ± SEM nighttime input resistance (Rin; 2256 ± 166 MΩ), compared to nontargeted shRNA-expressing SCN neurons (1143 ± 93 MΩ). Additional experiments revealed that acute knockdown of Kv4.1 significantly (p < 0.01) shortened, by â¼0.5 h, the circadian period of spontaneous electrical activity, clock gene expression and locomotor activity demonstrating a physiological role for Kv4.1-encoded IA channels in regulating circadian rhythms in neuronal excitability and behavior.
Assuntos
Ritmo Circadiano/fisiologia , Atividade Motora/fisiologia , Neurônios/metabolismo , Proteínas Circadianas Period/metabolismo , Canais de Potássio Shal/metabolismo , Núcleo Supraquiasmático/metabolismo , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Proteínas Circadianas Period/genética , Fotoperíodo , Interferência de RNA , Canais de Potássio Shal/genética , Técnicas de Cultura de TecidosRESUMO
The Kv channel interacting proteins (KChIPs) were identified in a yeast two hybrid screen using the N terminus of Kv4.3 as bait. Previous studies have demonstrated that KChIP2 associates with voltage-gated K(+) (Kv) pore-forming (alpha) subunits of the Kv4 subfamily and contributes to the formation of the rapidly inactivating and recovering Kv4-encoded cardiac transient outward K(+) channels, I(to,f). Here, we report that co-expression of KChIP2 (or KChIP1) also modulates the functional cell surface expression of Kv1.5-encoded K(+) channels in transiently transfected HEK-293 cells. In contrast to the effects of KChIP2 on Kv4 channels, however, co-expression of KChIP2 (or KChIP1) decreases Kv1.5-encoded K(+) currents. Although current densities are reduced, KChIP2 (or KChIP1) co-expression does not affect the time- or voltage-dependent properties of heterologously expressed Kv1.5-encoded K(+) currents. Immunohistochemical and cell surface biotinylation experiments demonstrate that KChIP2 reduces the cell surface expression of Kv1.5, likely by inhibiting forward trafficking from the endoplasmic reticulum. In addition, biochemical experiments reveal that KChIP2 co-immunoprecipitates with Kv1.5 (as well as Kv4.2/Kv4.3) from adult mouse ventricles, demonstrating that, similar to other Kv accessory subunits, KChIP2 is a multifunctional Kv channel accessory subunit. Taken together, the results here suggest that KChIP2 contributes to the formation of functional mouse ventricular (Kv1.5-encoded) I(K,slow1) channels as well, perhaps, as other Kv1.5-encoded K(+) currents, including I(Kur) (I(K,ultrarapid)), in human atria.