RESUMEN
CIB2 is a Ca2+- and Mg2+-binding protein essential for mechanoelectrical transduction (MET) by cochlear hair cells, but not by vestibular hair cells that co-express CIB2 and CIB3. Here, we show that in cochlear hair cells, CIB3 can functionally substitute for CIB2. Using X-ray crystallography, we demonstrate that CIB2 and CIB3 are structurally similar to KChIP proteins, auxiliary subunits of voltage-gated Kv4 channels. CIB2 and CIB3 bind to TMC1/2 through a domain in TMC1/2 flanked by transmembrane domains 2 and 3. The co-crystal structure of the CIB-binding domain in TMC1 with CIB3 reveals that interactions are mediated through a conserved CIB hydrophobic groove, similar to KChIP1 binding of Kv4. Functional studies in mice show that CIB2 regulates TMC1/2 localization and function in hair cells, processes that are affected by deafness-causing CIB2 mutations. We conclude that CIB2 and CIB3 are MET channel auxiliary subunits with striking similarity to Kv4 channel auxiliary subunits.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/fisiología , Células Ciliadas Auditivas/fisiología , Mecanotransducción Celular/fisiología , Animales , Cristalografía por Rayos X , Células HEK293 , Humanos , Proteínas de Interacción con los Canales Kv/química , Proteínas de Interacción con los Canales Kv/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Midbrain dopamine neurons (DANs) regulate various brain functions such as motor control and motivation. Alteration of spiking activities of these neurons could contribute to severe brain disorders including Parkinson's disease and depression. Previous studies showed important roles of somatodendritic voltage-gated K+ channels (Kv) of DANs in governing neuronal excitability and dopamine release. However, it remains largely unclear about the biophysical properties and the function of Kv channels distributed at DAN axons. We performed whole-cell recordings from the axons of DANs in acute mouse midbrain and striatal slices. We detected both rapidly activating/inactivating Kv current (i.e. A-current) and rapidly activating but slowly inactivating current (i.e. D-current) in DAN axons. Pharmacological experiments with channel blockers revealed that these currents are predominantly mediated by Kv1.4 and Kv1.2 subunits, respectively. Blocking these currents could substantially prolong axonal action potentials (APs) via a reduction of their repolarization slope. Together, our results show that Kv channels mediating A- and D-currents shape AP waveforms in midbrain DAN axons, through this regulation they may control dopamine release at the axonal terminals. Therefore, these axonal Kv channels could be drug targets for brain disorders with abnormal dopamine release.
Asunto(s)
Potenciales de Acción/fisiología , Axones/fisiología , Neuronas Dopaminérgicas/fisiología , Canal de Potasio Kv1.3/fisiología , Canal de Potasio Kv1.4/fisiología , Mesencéfalo/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Proteínas de Interacción con los Canales Kv/antagonistas & inhibidores , Proteínas de Interacción con los Canales Kv/fisiología , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.4/antagonistas & inhibidores , Masculino , Mesencéfalo/efectos de los fármacos , Ratones , Ratones Transgénicos , Bloqueadores de los Canales de Potasio/farmacologíaRESUMEN
The retinoblastoma protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related protein p130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and p130 knockout cells in primary human fibroblasts. Knockout of both p130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D-Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of p130 occurred during early G1, and led to the release of p130 and MuvB from E2F4 and decreased p130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and p130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.
Asunto(s)
Ciclo Celular/genética , Proliferación Celular/genética , Ciclina D/fisiología , Quinasa 4 Dependiente de la Ciclina/fisiología , Proteínas de Interacción con los Canales Kv/fisiología , Proteínas Represoras/fisiología , Proteína de Retinoblastoma/fisiología , Células A549 , Puntos de Control del Ciclo Celular/genética , Células Cultivadas , Regulación hacia Abajo/genética , Regulación de la Expresión Génica , Humanos , Recién Nacido , Masculino , Transducción de Señal/genéticaRESUMEN
DREAM (downstream regulatory element antagonist modulator), Calsenilin and KChIP3 (potassium channel interacting protein 3) belong to the neuronal calcium sensor (NCS) superfamily, which transduces the intracellular calcium signaling into a variety of activities. They are encoded by the same gene locus, but have distinct subcellular locations. DREAM was first found to interact with DRE (downstream regulatory element) site in the vicinity of the promoter of prodynorphin gene to suppress gene transcription. Calcium can disassemble this interaction by binding reversibly to DREAM protein on its four EF-hand motifs. Apart from having calcium dependent DRE site binding, DREAM can also interact with other transcription factors, such as cAMP responsive element binding protein (CREB), CREB-binding protein (CBP) and cAMP responsive element modulator (CREM), by this concerted actions, DREAM extends the gene pool under its control. DREAM is predominantly expressed in central nervous system with its highest level in cerebellum, and accumulating evidence demonstrated that DREAM might play important roles in pain sensitivity. Novel findings have shown that DREAM is also involved in learning and memory processes, Alzheimer's disease and stroke. This mini-review provides a brief introduction of its discovery history and protein structure properties, focusing on the mechanism of DREAM nuclear translocation and gene transcription regulation functions.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Interacción con los Canales Kv/fisiología , Proteínas Represoras/fisiología , Animales , Señalización del Calcio/fisiología , Humanos , Proteínas de Interacción con los Canales Kv/genética , Umbral del Dolor , Proteínas Represoras/genéticaRESUMEN
Pathologic electrical remodeling and attenuated cardiac contractility are featured characteristics of heart failure. Coinciding with these remodeling events is a loss of the K+ channel interacting protein, KChIP2. While, KChIP2 enhances the expression and stability of the Kv4 family of potassium channels, leading to a more pronounced transient outward K+ current, Ito,f, the guinea pig myocardium is unique in that Kv4 expression is absent, while KChIP2 expression is preserved, suggesting alternative consequences to KChIP2 loss. Therefore, KChIP2 was acutely silenced in isolated guinea pig myocytes, which led to significant reductions in the Ca2+ transient amplitude and prolongation of the transient duration. This change was reinforced by a decline in sarcomeric shortening. Notably, these results were unexpected when considering previous observations showing enhanced ICa,L and prolonged action potential duration following KChIP2 loss, suggesting a disruption of fundamental Ca2+ handling proteins. Evaluation of SERCA2a, phospholamban, RyR, and sodium calcium exchanger identified no change in protein expression. However, assessment of Ca2+ spark activity showed reduced spark frequency and prolonged Ca2+ decay following KChIP2 loss, suggesting an altered state of RyR activity. These changes were associated with a delocalization of the ryanodine receptor activator, presenilin, away from sarcomeric banding to more diffuse distribution, suggesting that RyR open probability are a target of KChIP2 loss mediated by a dissociation of presenilin. Typically, prolonged action potential duration and enhanced Ca2+ entry would augment cardiac contractility, but here we see KChIP2 fundamentally disrupts Ca2+ release events and compromises myocyte contraction. This novel role targeting presenilin localization and RyR activity reveals a significance for KChIP2 loss that reflects adverse remodeling observed in cardiac disease settings.
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Calcio/metabolismo , Proteínas de Interacción con los Canales Kv/fisiología , Células Musculares/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Cobayas , Inmunohistoquímica , Proteínas de Interacción con los Canales Kv/genéticaRESUMEN
Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain-derived neurotrophic factor in trigeminal ganglia. Genome-wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series "Pain".
Asunto(s)
Proteínas de Interacción con los Canales Kv/genética , Proteínas de Interacción con los Canales Kv/fisiología , Nocicepción/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Nervio Trigémino/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Catepsina L/metabolismo , Encefalinas/biosíntesis , Dolor Facial/fisiopatología , Hiperalgesia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Monoacilglicerol Lipasas/metabolismo , Estimulación Física , Precursores de Proteínas/biosíntesis , TranscriptomaRESUMEN
Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K(+) channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/enzimología , Proteínas de Interacción con los Canales Kv/fisiología , Animales , Cumarinas/metabolismo , Dipéptidos/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Proteínas de Drosophila/genética , Humanos , Canales de Potasio con Entrada de Voltaje/fisiología , Unión Proteica , Subunidades de Proteína/fisiología , Proteolisis , RatasRESUMEN
DREAM (also known as K(+) channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca(2+)-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer's disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb(3+), are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb(3+). We also observe that unlike Tb(3+)-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τaverage â¼ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb(3+) binds to at least three sites with high affinity (Kd = 1.8 µM in the presence of Ca(2+)) and displaces bound Ca(2+) through an entropically driven mechanism (ΔH â¼ 12 kcal mol(-1), and TΔS â¼ 22 kcal mol(-1)). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb(3+), like Ca(2+), triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca(2+) binding, Tb(3+) binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry-mass spectrometry reveal that replacement of Ca(2+) with Tb(3+) preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM-protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.
Asunto(s)
Proteínas de Interacción con los Canales Kv/química , Proteínas de Interacción con los Canales Kv/fisiología , Proteínas Represoras/química , Proteínas Represoras/fisiología , Terbio/química , Terbio/fisiología , Termodinámica , Secuencia de Aminoácidos , Animales , Ratones , Datos de Secuencia Molecular , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Gradients of the fast transient outward K+ current (Ito,f) contribute to heterogeneity of ventricular repolarization in a number of species. Cardiac Ito,f levels and gradients change notably with heart disease. Human cardiac Ito,f appears to be encoded by the Kv4.3 pore-forming α-subunit plus the auxiliary KChIP2 ß-subunit while mouse cardiac Ito,f requires Kv4.2 and Kv4.3 α-subunits plus KChIP2. Regional differences in cardiac Ito,f are associated with expression differences in Kv4.2 and KChIP2. Although Ito,f was reported to be absent in mouse ventricular cardiomyocytes lacking the Kv4.2 gene (Kv4.2-/-) when short depolarizing voltage pulses were used to activate voltage-gated K+ currents, in the present study, we showed that the use of long depolarization steps revealed a heteropodatoxin-sensitive Ito,f (at ~40% of the wild-type levels). Immunohistological studies further demonstrated membrane expression of Kv4.3 in Kv4.2-/- cardiomyocytes. Transmural Ito,f gradients across the left ventricular wall were reduced by ~3.5-fold in Kv4.2-/- heart, compared to wild-type. The Ito,f gradient in Kv4.2-/- hearts was associated with gradients in KChIP2 mRNA expression while in wild-type there was also a gradient in Kv4.2 expression. In conclusion, we found that Kv4.3-based Ito,f exists in the absence of Kv4.2, although with a reduced transmural gradient. Kv4.2-/- mice may be a useful animal model for studying Kv4.3-based Ito,f as observed in humans.
Asunto(s)
Potenciales de Acción/fisiología , Membrana Celular/fisiología , Miocitos Cardíacos/fisiología , Canales de Potasio Shal/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Expresión Génica , Proteínas de Interacción con los Canales Kv/genética , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas de Interacción con los Canales Kv/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , Venenos de Araña/farmacologíaRESUMEN
RATIONALE: Chronic alcohol-induced cognitive impairments and maladaptive plasticity of glutamatergic synapses are well-documented. However, it is unknown if prolonged alcohol exposure affects dendritic signaling that may underlie hippocampal dysfunction in alcoholics. Back-propagation of action potentials (bAPs) into apical dendrites of hippocampal neurons provides distance-dependent signals that modulate dendritic and synaptic plasticity. The amplitude of bAPs decreases with distance from the soma that is thought to reflect an increase in the density of Kv4.2 channels toward distal dendrites. OBJECTIVE: The aim of this study was to quantify changes in hippocampal Kv4.2 channel function and expression using electrophysiology, Ca(2+) imaging, and western blot analyses in a well-characterized in vitro model of chronic alcohol exposure. RESULTS: Chronic alcohol exposure significantly decreased expression of Kv4.2 channels and KChIP3 in hippocampus. This reduction was associated with an attenuation of macroscopic A-type K(+) currents in CA1 neurons. Chronic alcohol exposure increased bAP-evoked Ca(2+) transients in the distal apical dendrites of CA1 pyramidal neurons. The enhanced bAP-evoked Ca(2+) transients induced by chronic alcohol exposure were not related to synaptic targeting of N-methyl-D-aspartate (NMDA) receptors or morphological adaptations in apical dendritic arborization. CONCLUSIONS: These data suggest that chronic alcohol-induced decreases in Kv4.2 channel function possibly mediated by a downregulation of KChIP3 drive the elevated bAP-associated Ca(2+) transients in distal apical dendrites. Alcohol-induced enhancement of bAPs may affect metaplasticity and signal integration in apical dendrites of hippocampal neurons leading to alterations in hippocampal function.
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Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Alcoholismo/fisiopatología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Dendritas/efectos de los fármacos , Dendritas/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Proteínas de Interacción con los Canales Kv/efectos de los fármacos , Proteínas de Interacción con los Canales Kv/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Canales de Potasio Shal/efectos de los fármacos , Canales de Potasio Shal/fisiología , Animales , Calcio/metabolismo , Femenino , Células Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismoRESUMEN
In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules.
Asunto(s)
Potenciales de Acción/fisiología , Señalización del Calcio/fisiología , Dendritas/fisiología , Proteínas de Interacción con los Canales Kv/fisiología , Células de Purkinje/fisiología , Transducción de Señal/fisiología , Animales , Dendritas/ultraestructura , Activación del Canal Iónico/fisiología , Ratones , Técnicas de Cultivo de Órganos , Células de Purkinje/ultraestructura , Ratas , Ratas WistarRESUMEN
In the field of molecular and cellular neuroscience, it is not a trivial task to see the forest for the trees, where numerous, and seemingly independent, molecules often work in concert to control critical steps of synaptic plasticity and signalling. Here, we will first summarize our current knowledge on essential activity-dependent transcription factors (TFs) such as CREB, MEF2, Npas4 and SRF, then examine how various transcription cofactors (TcoFs) also contribute to defining the transcriptional outputs during learning and memory. This review finally attempts a provisory synthesis that sheds new light on some of the emerging principles of neuronal circuit dynamics driven by activity-regulated gene transcription to help better understand the intricate relationship between activity-dependent gene expression and cognitive behavior.
Asunto(s)
Cognición/fisiología , Regulación de la Expresión Génica/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteína de Unión a CREB/fisiología , Humanos , Proteínas de Interacción con los Canales Kv/fisiología , Aprendizaje/fisiología , Factores de Transcripción MEF2/fisiología , Memoria/fisiología , Proteína 2 de Unión a Metil-CpG/fisiología , Proteínas Represoras/fisiologíaRESUMEN
In the mammalian brain, information encoding and storage have been explained by revealing the cellular and molecular mechanisms of synaptic plasticity at various levels in the central nervous system, including the hippocampus and the cerebral cortices. The modulatory mechanisms of synaptic excitability that are correlated with neuronal tasks are fundamental factors for synaptic plasticity, and they are dependent on intracellular Ca(2+)-mediated signaling. In the present review, the A-type K(+) (IA) channel, one of the voltage-dependent cation channels, is considered as a key player in the modulation of Ca(2+) influx through synaptic NMDA receptors and their correlated signaling pathways. The cellular functions of IA channels indicate that they possibly play as integral parts of synaptic and somatic complexes, completing the initiation and stabilization of memory.
Asunto(s)
Hipocampo/citología , Proteínas de Interacción con los Canales Kv/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/metabolismo , Animales , Neuronas/citologíaRESUMEN
Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the transient outward K(+) current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2(-/-) mice. Echocardiography was performed before and every 2 weeks after the operation. Ten weeks post-surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC-operated WT and KChIP2(-/-) mice. Ventricular protein expression of KChIP2 was reduced by 70% after 10 weeks TAC in WT mice. The amplitudes of the J and T waves were enlarged in KChIP2(-/-) control mice. Ventricular effective refractory period, RR, QRS and QT intervals were longer in mice with HF compared to sham-operated mice of either genotype. Pacing-induced ventricular tachycardia (VT) was observed in 5/10 sham-operated WT mice compared with 2/10 HF WT mice with HF. Interestingly, and contrary to previously published data, sham-operated KChIP2(-/-) mice were resistant to pacing-induced VT resulting in only 1/10 inducible mice. KChIP2(-/-) with HF mice had similar low vulnerability to inducible VT (1/9). Our results suggest that although KChIP2 is downregulated in HF, it is not orchestrating the development of HF. Moreover, KChIP2 affects ventricular repolarization and lowers arrhythmia susceptibility. Hence, downregulation of KChIP2 expression in HF may be antiarrhythmic in mice via reduction of the fast transient outward K(+) current.
Asunto(s)
Insuficiencia Cardíaca/fisiopatología , Proteínas de Interacción con los Canales Kv/fisiología , Animales , Arritmias Cardíacas/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
RATIONALE: A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current (I(to)) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF I(to) and that its overexpression might specifically alter PF I(to) properties and repolarization. OBJECTIVE: To assess the potential role of DPP6 in PF I(to). METHODS AND RESULTS: Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle I(to) had similar density, but PF I(to) differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, I(to) density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K(+)-channel interacting ß-subunit K(+)-channel interacting protein type-2, essential for normal expression of I(to) in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small I(to); I(to) amplitude was greatly enhanced by coexpression with K(+)-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K(+)-channel interacting protein type-2 failed to alter I(to) compared with Kv4.3/K(+)-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF I(to) composition) greatly enhanced I(to) compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that I(to) enhancement can greatly accelerate PF repolarization. CONCLUSIONS: These results point to a previously unknown central role of DPP6 in PF I(to), with DPP6 gain of function selectively enhancing PF current, and suggest that a DPP6-mediated PF early-repolarization syndrome might be a novel molecular paradigm for some forms of idiopathic ventricular fibrillation.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/fisiología , Proteínas de Interacción con los Canales Kv/fisiología , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio/fisiología , Ramos Subendocárdicos/fisiología , Canales de Potasio Shal/fisiología , Fibrilación Ventricular/fisiopatología , Adulto , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Modelos Animales de Enfermedad , Perros , Femenino , Técnicas de Silenciamiento del Gen , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Técnicas In Vitro , Proteínas de Interacción con los Canales Kv/efectos de los fármacos , Proteínas de Interacción con los Canales Kv/genética , Masculino , Persona de Mediana Edad , Modelos Teóricos , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Ramos Subendocárdicos/patología , Canales de Potasio Shal/efectos de los fármacos , Canales de Potasio Shal/genética , Tetraetilamonio/farmacología , TransfecciónRESUMEN
BACKGROUND: The necessity for, or redundancy of, distinctive KChIP proteins is not known. RESULTS: Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. CONCLUSION: KChIP2 is essential for homeostasis in hippocampal neurons. SIGNIFICANCE: Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K(+) current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2(-/-) mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2(-/-) neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2(-/-) mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2(-/-) mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.
Asunto(s)
Potenciales de Acción , Homeostasis , Proteínas de Interacción con los Canales Kv/fisiología , Subunidades de Proteína/fisiología , Amígdala del Cerebelo/patología , Amígdala del Cerebelo/fisiopatología , Animales , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/fisiopatología , Células Cultivadas , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores , Excitación Neurológica , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Células Piramidales/metabolismo , Células Piramidales/fisiología , ConvulsionesRESUMEN
A-type K(+) channels are crucial in controlling neuronal excitability, and their regulation in sensory neurons may alter pain sensation. In this study, we identified the functional role of cobrotoxin, the short-chain α-neurotoxin isolated from Naja atra venom, which acts in the regulation of the transient A-type K(+) currents (IA) and membrane excitability in dorsal root ganglion (DRG) neurons via the activation of the muscarinic M3 receptor (M3R). Our results showed that cobrotoxin increased IA in a concentration-dependent manner, whereas the sustained delayed rectifier K(+) currents (IDR) were not affected. Cobrotoxin did not affect the activation of IA markedly, however, it shifted the inactivation curve significantly in the depolarizing direction. The cobrotoxin-induced IA response was blocked by the M3R-selective antagonists DAU-5884 and 4-DAMP. An siRNA targeting the M3R in small DRG neurons abolished the cobrotoxin-induced IA increase. In addition, dialysis of the cells with the novel protein kinase C-delta isoform (PKC-δ) inhibitor δv1-1 or an siRNA targeting PKC-δ abolished the cobrotoxin-induced IA response, whereas inhibition of PKA or classic PKC activity elicited no such effects. Moreover, we observed a significant decrease in the firing rate of the neuronal action potential induced by M3R activation. Pretreatment of the cells with 4-aminopyridine, a selective blocker of IA, abolished this effect. Taken together, these results suggest that the short-chain cobrotoxin selectively enhances IA via a novel PKC-δ-dependent pathway. This effect occurred via the activation of M3R and might contribute to its neuronal hypoexcitability in small DRG neurons.
Asunto(s)
Membrana Celular/fisiología , Proteínas Neurotóxicas de Elápidos/farmacología , Ganglios Espinales/fisiología , Proteínas de Interacción con los Canales Kv/fisiología , Neuronas/fisiología , Proteína Quinasa C-delta/fisiología , Animales , Membrana Celular/efectos de los fármacos , Ganglios Espinales/citología , Técnicas de Silenciamiento del Gen , Técnicas In Vitro , Ratones , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Proteína Quinasa C-delta/genética , Receptor Muscarínico M3/fisiologíaRESUMEN
We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (I(SA)) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary ß-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the I(SA) component in hippocampal neurons was reduced in the presence of 20 µM NS5806. I(SA) decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native I(SA) and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action.
Asunto(s)
Hipocampo/metabolismo , Compuestos de Fenilurea/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Shal/efectos de los fármacos , Tetrazoles/farmacología , Animales , Células Cultivadas , Interpretación Estadística de Datos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/efectos de los fármacos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/fisiología , Células HEK293 , Hipocampo/efectos de los fármacos , Humanos , Técnicas In Vitro , Cinética , Proteínas de Interacción con los Canales Kv/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.