RESUMEN
AIMS: Small conductance Ca(2+)-activated K(+) channels (K(Ca)2 or SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca(2+) (Ca(i)²âº) with membrane potentials. We have recently reported the functional expression of SK channels in human and mouse cardiac myocytes. Additionally, we have found that the channel is highly expressed in atria compared with the ventricular myocytes. We demonstrated that human cardiac myocytes expressed all three members of SK channels (SK1, 2, and 3); moreover, the different members are capable of forming heteromultimers. Here, we directly tested the contribution of SK3 to the overall repolarization of atrial action potentials. METHODS AND RESULTS: We took advantage of a mouse model with site-specific insertion of a tetracycline-based genetic switch in the 5' untranslated region of the KCNN3 (SK3 channel) gene (SK3(T/T)). The gene-targeted animals overexpress the SK3 channel without interfering with the normal profile of SK3 expression. Whole-cell, patch-clamp techniques show a significant shortening of the action potential duration mainly at 90% repolarization (APD90) in atrial myocytes from the homozygous SK3(T/T) animals. Conversely, treatment with dietary doxycycline results in a significant prolongation of APD90 in atrial myocytes from SK3(T/T) animals. We further demonstrate that the shortening of APDs in SK3 overexpression mice predisposes the animals to inducible atrial arrhythmias. CONCLUSION: SK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes.
Asunto(s)
Función Atrial , Miocitos Cardíacos/metabolismo , Potasio/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Potenciales de Acción , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Electrocardiografía , Predisposición Genética a la Enfermedad , Atrios Cardíacos/diagnóstico por imagen , Atrios Cardíacos/metabolismo , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Técnicas de Placa-Clamp , Fenotipo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Factores de Tiempo , UltrasonografíaRESUMEN
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.
Asunto(s)
Nivel de Alerta/fisiología , Fases del Sueño/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Núcleos Talámicos/fisiología , Potenciales de Acción , Animales , Umbral Auditivo , Células Cultivadas/fisiología , Electroencefalografía , Femenino , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Polisomnografía , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/biosíntesis , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Organismos Libres de Patógenos Específicos , Núcleos Talámicos/citología , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Órgano Vomeronasal/fisiología , Agresión/fisiología , Animales , Conducta Animal/fisiología , Dendritas/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Moco/metabolismo , Técnicas de Placa-Clamp , Potasio/farmacología , Conducta Sexual Animal/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Estimulación Química , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/fisiología , Orina/fisiologíaRESUMEN
In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca(2+)-activated K(+) channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg(2+) block that reduces Ca(2+) influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.
Asunto(s)
Isquemia Encefálica/patología , Neuronas/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Animales , Conducta Animal/fisiología , Bencimidazoles/farmacología , Isquemia Encefálica/psicología , Región CA1 Hipocampal/patología , Reanimación Cardiopulmonar , Muerte Celular , Paro Cardíaco/complicaciones , Paro Cardíaco/patología , Inmunohistoquímica , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Inmunoelectrónica , Actividad Motora/fisiología , Neuronas/patología , Técnicas de Placa-Clamp , Células Piramidales/patología , Reconocimiento en Psicología/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Sinapsis/fisiología , Sinapsis/ultraestructuraRESUMEN
SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse hippocampal area CA1 pyramidal neurons and influence synaptic responses, plasticity and learning. The Sk2 gene (also known as Kcnn2) encodes two isoforms that differ only in the length of their N-terminal domains. SK2-long (SK2-L) and SK2-short (SK2-S) are coexpressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-S only mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to excitatory postsynaptic potentials was absent in SK2-S only mice and was restored by SK2-L re-expression. Blocking SK channels increased the amount of long-term potentiation induced in area CA1 in slices from wild-type mice but had no effect in slices from SK2-S only mice. Furthermore, SK2-S only mice outperformed wild-type mice in the novel object recognition task. These results indicate that SK2-L directs synaptic SK2-containing channel expression and is important for normal synaptic signaling, plasticity and learning.
Asunto(s)
Región CA1 Hipocampal/citología , Células Piramidales/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Sinapsis/metabolismo , Animales , Potenciales Postsinápticos Excitadores/genética , Potenciación a Largo Plazo/genética , Ratones , Ratones Noqueados , Densidad Postsináptica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/deficiencia , Sinapsis/genéticaRESUMEN
BACKGROUND: It has been proposed that activation of endothelial SK3 (K(Ca)2.3) and IK1 (K(Ca)3.1) K+ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. METHODS AND RESULTS: Expression and function of endothelial SK3 and IK1 in IK1(-/-)/SK3(T/T) mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1(-/-)/SK3(T/T) (+doxycycline) mice abolished endothelial K(Ca) currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1(-/-)/SK3(T/T) mice partially restored EDHF- and nitric oxide-mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1(+/+)/SK3(T/T) (+doxycycline) mice to normotensive levels. CONCLUSIONS: Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial K(Ca) channels may represent novel therapeutic targets for the treatment of hypertension.
Asunto(s)
Factores Biológicos/fisiología , Hipertensión/etiología , Vasodilatación , Animales , Factores Biológicos/metabolismo , Presión Sanguínea/fisiología , Calcio/metabolismo , Potenciales de la Membrana , Ratones , Ratones Noqueados , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/fisiología , Canales de Potasio Shaw/deficiencia , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/deficienciaRESUMEN
Small conductance Ca(2+)-activated K(+) channels (SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca(2+) (Ca(2+)(i)) with membrane potential. We have recently reported the functional existence of SK2 channels in human and mouse cardiac myocytes. Moreover, we have found that the channel is predominantly expressed in atria compared to the ventricular myocytes. We hypothesize that knockout of SK2 channels may be sufficient to disrupt the intricate balance of the inward and outward currents during repolarization in atrial myocytes. We further predict that knockout of SK2 channels may predispose the atria to tachy-arrhythmias due to the fact that the late phase of the cardiac action potential is highly susceptible to aberrant excitation. We take advantage of a mouse model with genetic knockout of the SK2 channel gene. In vivo and in vitro electrophysiological studies were performed to probe the functional roles of SK2 channels in the heart. Whole-cell patch-clamp techniques show a significant prolongation of the action potential duration prominently in late cardiac repolarization in atrial myocytes from the heterozygous and homozygous null mutant animals. Moreover, in vivo electrophysiological recordings show inducible atrial fibrillation in the null mutant mice but not wild-type animals. No ventricular arrhythmias are detected in the null mutant mice or wild-type animals. In summary, our data support the important functional roles of SK2 channels in cardiac repolarization in atrial myocytes. Genetic knockout of the SK2 channels results in the delay in cardiac repolarization and atrial arrhythmias.
Asunto(s)
Potenciales de Acción/genética , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Miocitos Cardíacos/patología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/deficiencia , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Cochlear hair cells use SK2 currents to shape responses to cholinergic efferent feedback from the brain. Using SK2(-/-) mice, we demonstrate that, in addition to their previously defined role in modulating hair cell membrane potentials, SK2 channels are necessary for long-term survival of olivocochlear fibers and synapses. Loss of the SK2 gene also results in loss of electrically driven olivocochlear effects in vivo, and down regulation of ryanodine receptors involved in calcium-induced calcium release, the main inducer of nAChR evoked SK2 activity. Generation of double-null mice lacking both the alpha10 nAChR gene, loss of which results in hypertrophied olivocochlear terminals, and the SK2 gene, recapitulates the SK2(-/-) synaptic phenotype and gene expression, and also leads to down regulation of alpha9 nAChR gene expression. The data suggest a hierarchy of activity necessary to maintain early olivocochlear synapses at their targets, with SK2 serving an epistatic, upstream, role to the nAChRs.
Asunto(s)
Supervivencia Celular/fisiología , Cóclea/citología , Cóclea/inervación , Vías Eferentes/anatomía & histología , Células Ciliadas Auditivas Externas/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Sinapsis/metabolismo , Animales , Cóclea/fisiología , Vías Eferentes/fisiología , Células Ciliadas Auditivas Externas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Núcleo Olivar/anatomía & histología , Núcleo Olivar/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Sinaptofisina/metabolismoRESUMEN
T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.
Asunto(s)
Relojes Biológicos/fisiología , Dendritas/fisiología , Núcleos Talámicos de la Línea Media/citología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/fisiología , Sueño/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Anestésicos Locales/farmacología , Animales , Animales Recién Nacidos , Apamina/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Dendritas/ultraestructura , Estimulación Eléctrica/métodos , Electroencefalografía/métodos , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Mibefradil/farmacología , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Técnicas de Placa-Clamp , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/deficiencia , Tetrodotoxina/farmacología , Caminata/fisiologíaRESUMEN
Hippocampal-dependent synaptic plasticity and memory are modulated by apamin-sensitive small conductance Ca2+-activated K+ (SK) channels. Transgenic mice overexpressing SK2 channels (SK2+/T mice) exhibit marked deficits in hippocampal memory and synaptic plasticity, as previously reported. Here, we examined whether SK2 overexpression affects the encoding or retention of contextual memory. Compared with wild-type littermates, SK2+/T mice exhibited significantly less context-dependent freezing 10 min and 24 h after conditioning. Interestingly, this contextual memory impairment was eliminated if SK2+/T mice were permitted longer pre-exposure to the conditioning chamber. These data support converging evidence that SK2 channels restrict the encoding of hippocampal memory.
Asunto(s)
Trastornos de la Memoria/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Animales , Miedo/fisiología , Regulación de la Expresión Génica , Genotipo , Hipocampo/fisiopatología , Ratones , Sinapsis/fisiologíaRESUMEN
Since the first description of the anatomical atrioventricular nodes (AVNs), a large number of studies have provided insights into the heterogeneity of the structure as well as a repertoire of ion channel proteins that govern this complex conduction pathway between the atria and ventricles. These studies have revealed the intricate organization of multiple nodal and nodal-like myocytes contributing to the unique electrophysiology of the AVN in health and diseases. On the other hand, information regarding the contribution of specific ion channels to the function of the AVN remains incomplete. We reason that the identification of AVN-specific ion channels may provide a more direct and rational design of therapeutic target in the control of AVN conduction in atrial flutter/fibrillation, one of the most common arrhythmias seen clinically. In this study, we took advantage of 2 genetically altered mouse models with overexpression or null mutation of 1 of a small conductance Ca2+-activated K+ channel isoform, SK2 channel, and demonstrated robust phenotypes of AVN dysfunction in these experimental models. Overexpression of SK2 channels results in the shortening of the spontaneous action potentials of the AVN cells and an increase in the firing frequency. On the other hand, ablation of the SK2 channel results in the opposite effects on the spontaneous action potentials of the AVN. Furthermore, we directly documented the expression of SK2 channel in mouse AVN using multiple techniques. The new insights may have important implications in providing novel drug targets for the modification of AVN conduction in the treatment of atrial arrhythmias.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Nodo Atrioventricular/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Potenciales de Acción/fisiología , Animales , Arritmias Cardíacas/genética , Nodo Atrioventricular/citología , Electrocardiografía , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Ratones , Ratones Transgénicos , Microscopía Confocal , Técnicas de Placa-Clamp , Nodo Sinoatrial/citología , Nodo Sinoatrial/fisiologíaRESUMEN
Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specific isoform of small conductance Ca(2+)-activated K(+) (SK) channel, SK3, promotes feedback regulation of myometrial Ca(2+) and hence relaxation of the uterus. To determine the specific functional impact of SK3 channels, we assessed isometric contractions of uterine strips from genetically altered mice (SK3(T/T)), in which SK3 is overexpressed and can be suppressed by oral administration of doxycycline (SK3(T/T)+Dox). We found SK3 protein in mouse myometrium, and this expression was substantially higher in SK3(T/T) mice and lower in SK3(T/T)+Dox mice compared with wild-type (WT) controls. Sustained contractions elicited by 60 mM KCl were not different among SK3(T/T), SK3(T/T)+Dox, and WT mice. However, the rate of onset and magnitude of spontaneously occurring phasic contractions was muted significantly in isolated uterine strips from SK3(T/T) mice compared with those from WT mice. These spontaneous contractions were augmented greatly by blockade of SK channels with apamin or by suppression of SK3 expression. Phasic but not tonic contraction in response to oxytocin was depressed in uterine strips from SK3(T/T) mice, whereas suppression of SK3 channel expression or treatment with apamin promoted the predominance of large coordinated phasic events over tone. Spontaneous contractions and the phasic component of oxytocin contractions were blocked by nifedipine but not by cyclopiazonic acid. Our findings suggest that SK3 channels play an important role in regulating uterine function by limiting influx through L-type Ca(2+) channels and disrupting the development of concerted phasic contractile events.
Asunto(s)
Calcio/fisiología , Miometrio/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Animales , Apamina/farmacología , Doxiciclina/farmacología , Femenino , Técnicas In Vitro , Indoles/farmacología , Contracción Isométrica , Ratones , Ratones Endogámicos C57BL , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Nifedipino/farmacología , Oxitocina/farmacología , Periodicidad , Embarazo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Contracción Uterina , Vasodilatadores/farmacologíaRESUMEN
Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.
Asunto(s)
Hipocampo/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Plasticidad Neuronal/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Sinapsis/fisiología , Animales , Cartilla de ADN , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Heterocigoto , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genéticaRESUMEN
Supraoptic nucleus (SON) neurons possess a prominent afterhyperpolarization (AHP) that contributes to spike patterning. This AHP is probably underlain by a small-conductance, CA2+-dependent, K+ type 3 (SK3) channel. To determine the distribution of SK3 channels within the SON, we used immunocytochemistry in rats and in transgenic mice with a regulatory cassette on the SK3 gene, allowing regulated expression with dietary doxycycline (DOX). In rats and wild-type mice, SK3 immunostaining revealed an intense lacy network surrounding SON neurons, with weak staining in neuronal somata and dendrites. In untreated, conditional SK3 knockout mice, SK3 was overexpressed, but the pericellular pattern in the SON was similar to that of rats. DOX-treated transgenic mice exhibited no SK3 staining in the SON. Double staining for oxytocin or vasopressin neurons revealed weak co-localization with SK3 but strong staining surrounding each neuron type. Electron microscopy showed that SK3-like immunoreactivity was intense between neuronal somata and dendrites, in apparent glial processes, but weak in neurons. This was confirmed by using confocal microscopy and double staining for glial fibrillary acidic protein (GFAP) and SK3: many GFAP-positive processes in the SON, and in the ventral dendritic/glial lamina, were shown to contain SK3-like immunoreactivity. These studies suggest a prominent role of SK3 channels in astrocytes. Given the marked plasticity in glial/neuronal relationships, as well as studies suggesting that astrocytes in the central nervous system can generate prominent CA2+ transients to various stimuli, a CA2+-dependent K+ channel may help SON astrocytes with K+ buffering whenever astrocyte intracellular CA2+ is increased.
Asunto(s)
Astrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Canales de Potasio Calcio-Activados/metabolismo , Núcleo Supraóptico/citología , Animales , Astrocitos/ultraestructura , Doxiciclina/administración & dosificación , Femenino , Alimentos Formulados , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Transgénicos , Microscopía Inmunoelectrónica/métodos , Neurofisinas/metabolismo , Canales de Potasio Calcio-Activados/genética , Ratas , Ratas Sprague-Dawley , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Sustancia Negra/metabolismoRESUMEN
Small conductance calcium-activated potassium channels link elevations of intracellular calcium ions to membrane potential, exerting a hyperpolarizing influence when activated. The consequences of SK channel activity have been revealed by the specific blocker apamin, a peptide toxin from honeybee venom. Recent studies have revealed unexpected roles for SK channels in fine-tuning intrinsic cell firing properties and in responsiveness to synaptic input. They have also identified specific roles for different SK channel subtypes. A host of Ca2+ sources, including distinct subtypes of voltage-dependent calcium channels, intracellular Ca2+ stores and Ca2+-permeable ionotropic neurotransmitter receptors, activate SK channels. The macromolecular complex in which the Ca2+ source, SK channels and various modulators are assembled determines the kinetics and consequences of SK channel activation.
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Relojes Biológicos/fisiología , Encéfalo/fisiología , Modelos Neurológicos , Canales de Potasio Calcio-Activados/metabolismo , Sinapsis/metabolismo , Animales , Calcio/metabolismo , Potenciales de la Membrana/fisiología , Neuronas/fisiologíaRESUMEN
The SK2 subtype of small conductance Ca2+-activated K+ channels is widely distributed throughout the central nervous system and modulates neuronal excitability by contributing to the afterhyperpolarization that follows an action potential. Western blots of brain membrane proteins prepared from wild type and SK2-null mice reveal two isoforms of SK2, a 49-kDa band corresponding to the previously reported SK2 protein (SK2-S) and a novel 78-kDa form. Complementary DNA clones from brain and Western blots probed with an antibody specific for the longer form, SK2-L, identified the larger molecular weight isoform as an N-terminally extended SK2 protein. The N-terminal extension of SK2-L is cysteine-rich and mediates disulfide bond formation between SK2-L subunits or with heterologous proteins. Immunohistochemistry revealed that in brain SK2-L and SK2-S are expressed in similar but not identical patterns. Heterologous expression of SK2-L results in functional homomeric channels with Ca2+ sensitivity similar to that of SK2-S, consistent with their shared core and intracellular C-terminal domains. In contrast to the diffuse, uniform surface distribution of SK2-S, SK2-L channels cluster into sharply defined, distinct puncta suggesting that the extended cysteine-rich N-terminal domain mediates this process. Immunoprecipitations from transfected cells and mouse brain demonstrate that SK2-L co-assembles with the other SK subunits. Taken together, the results show that the SK2 gene encodes two subunit proteins and suggest that native SK2-L subunits may preferentially partition into heteromeric channel complexes with other SK subunits.
Asunto(s)
Encéfalo/metabolismo , Canales de Potasio Calcio-Activados/química , Canales de Potasio Calcio-Activados/fisiología , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Células COS , Calcio/metabolismo , Membrana Celular/metabolismo , Corteza Cerebral/metabolismo , Cricetinae , Cisteína/química , ADN Complementario/metabolismo , Disulfuros , Relación Dosis-Respuesta a Droga , Electrofisiología , Hipocampo/metabolismo , Inmunohistoquímica , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , TransfecciónRESUMEN
Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+ (SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP.
Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo , Neuronas/fisiología , Canales de Potasio Calcio-Activados/fisiología , Animales , Apamina/farmacología , Western Blotting , Relación Dosis-Respuesta a Droga , Potenciales Evocados , Hipocampo/metabolismo , Hibridación in Situ , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Canales de Potasio Calcio-Activados/genética , ARN Mensajero/aislamiento & purificaciónRESUMEN
Small conductance Ca(2+)-activated K(+) channels (SK channels) contribute to the long lasting afterhyperpolarization (AHP) that follows an action potential in many central neurones. The biophysical and pharmacological attributes of cloned SK channels strongly suggest that one or more of them underlie the medium component of the AHP that regulates interspike interval and plays an important role in setting tonic firing frequency. The cloned SK channels comprise a distinct subfamily of K(+) channels. Heterologously expressed SK channels recapitulate the biophysical and pharmacological hallmarks of native SK channels, being gated solely by intracellular Ca(2+) ions with no voltage dependence to their gating, small unitary conductance values and sensitivity to the bee venom peptide toxin, apamin. Molecular, biochemical and electrophysiological studies have revealed that Ca(2+) gating in SK channels is due to heteromeric assembly of the SK alpha pore-forming subunits with calmodulin (CaM). Ca(2+) binding to the N-terminal E-F hands of CaM is responsible for SK channel gating. Crystallographic studies suggest that SK channels gate as a dimer-of-dimers, and that the physical gate of SK channels resides at or near the selectivity filter of the channels. In addition, Ca(2+)-independent interactions between the SK channel alpha subunits and CaM are necessary for proper membrane trafficking.
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Calmodulina/fisiología , Canales de Potasio Calcio-Activados/fisiología , Canales de Potasio/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/química , Calcio/fisiología , Calmodulina/química , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales de Potasio/química , Canales de Potasio Calcio-Activados/química , Canales de Potasio de Pequeña Conductancia Activados por el CalcioRESUMEN
Small conductance, calcium-activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4-fold in isolated myocytes from SK3T/T mice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1-3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3T/T had significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non-voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non-voiding contractions in vivo. Activation of these channels may be a therapeutic approach for management of non-voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.
Asunto(s)
Músculo Liso/fisiología , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Enfermedades de la Vejiga Urinaria/fisiopatología , Vejiga Urinaria/fisiología , Animales , Apamina/farmacología , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Hipertonía Muscular/fisiopatología , Técnicas de Placa-Clamp , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , OrinaRESUMEN
The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca2+-activated K+ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure- and phenylephrine-induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure.