RESUMEN
The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.
Asunto(s)
Potenciales de Acción , Eliminación de Gen , Neuronas/fisiología , Fenotipo , Convulsiones/genética , Canales de Potasio Shab/metabolismo , Animales , Convulsivantes/farmacología , Flurotilo/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Potenciación a Largo Plazo , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Pilocarpina/farmacología , Convulsiones/fisiopatología , Canales de Potasio Shab/genéticaRESUMEN
Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the CNS, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca(2+), which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocin-secreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects.
Asunto(s)
Encéfalo/metabolismo , Canales Iónicos/metabolismo , Neuronas/metabolismo , Oxitocina/análogos & derivados , Hipófisis/metabolismo , Terminales Presinápticos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Anticuerpos Monoclonales , Encéfalo/ultraestructura , Calcio/metabolismo , Permeabilidad de la Membrana Celular , Reacciones Cruzadas , Femenino , Canales Iónicos/inmunología , Neuronas/ultraestructura , Oocitos/metabolismo , Oxitocina/metabolismo , Hipófisis/ultraestructura , Xenopus , Proteínas de Pez Cebra/inmunologíaRESUMEN
Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of 2-3 weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.
Asunto(s)
Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/patología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Pilocarpina , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Hipocampo/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Interacción con los Canales Kv/metabolismo , Canal de Potasio Kv1.4/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/clasificación , Neuronas/citología , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shal/metabolismo , Factores de TiempoRESUMEN
Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and potassium channel interacting protein (KChIP) 1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal Ammon's horn (CA1) neurons. Immunohistochemistry was performed on 40 mum rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with a unique dendritic A-type channel complement and ability to control BAPs.
Asunto(s)
Dendritas/metabolismo , Hipocampo/metabolismo , Interneuronas/metabolismo , Canales de Potasio Shal/genética , Animales , Calbindina 2 , Calbindinas , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Activación del Canal Iónico/fisiología , Proteínas de Interacción con los Canales Kv/biosíntesis , Proteínas de Interacción con los Canales Kv/genética , Parvalbúminas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/metabolismo , Canales de Potasio Shal/biosíntesis , Somatostatina/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Voltage-gated K(+) channels are key regulators of neuronal excitability. The Kv2.1 voltage-gated K(+) channel is the major delayed rectifier K(+) channel expressed in most central neurons, where it exists as a highly phosphorylated protein. Kv2.1 plays a critical role in homoeostatic regulation of intrinsic neuronal excitability through its activity- and calcineurin-dependent dephosphorylation. Here, we review studies leading to the identification and functional characterization of in vivo Kv2.1 phosphorylation sites, a subset of which contribute to graded modulation of voltage-dependent gating. These findings show that distinct developmental-, cell- and state-specific regulation of phosphorylation at specific sites confers a diversity of functions on Kv2.1 that is critical to its role as a regulator of intrinsic neuronal excitability.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio Shab/fisiología , Línea Celular , Humanos , Neuronas/fisiología , Fosforilación , Canales de Potasio Shab/química , Canales de Potasio Shab/metabolismo , Espectrometría de Masas en TándemRESUMEN
The challenge of accurately predicting human clinical outcome based on preclinical data has led to a high failure rate of compounds in human clinical trials. A series of methods are described by which biosimulation can address these challenges and guide the design and evaluation of experimental and clinical protocols. Early compound development often proceeds on the basis of preclinical data from animal models. The systematic evaluation possible in a simulation can assist in the critical step of translating the preclinical outcomes to human physiology. Later in the process, clinical trials definitively establish a therapy's beneficial effects, as well as any adverse side effects. Biosimulation allows for the optimal design of clinical trials to ensure that key issues are addressed effectively and efficiently, and in doing so, improves the success rate of the trials.
Asunto(s)
Bioensayo/métodos , Ensayos Clínicos como Asunto/métodos , Diabetes Mellitus/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Glucógeno/metabolismo , Insulina/administración & dosificación , Hígado/metabolismo , Animales , Simulación por Computador , Diabetes Mellitus/tratamiento farmacológico , Modelos Animales de Enfermedad , Diseño de Fármacos , Industria Farmacéutica/métodos , Humanos , Hígado/efectos de los fármacos , Masculino , Modelos Biológicos , Ratas , Ratas Wistar , Proyectos de Investigación , Especificidad de la EspecieRESUMEN
Dendritic excitability is modulated by the highly variable spatial and temporal expression pattern of voltage-dependent potassium channels. Somatodendritic Kv2.1 channels contribute a major component of delayed rectifier potassium current in cultured hippocampal neurons, where Kv2.1 is localized to large clusters on the soma and proximal dendrites. Here we found that dramatic differences exist in the clustering of endogenous Kv2.1 in cultured rat hippocampal GABAergic interneurons and glutamatergic pyramidal neurons. Studies on neurons developing in culture revealed that while a similar sequence of Kv2.1 localization and clustering occurred in both cell types, the process was temporally delayed in pyramidal cells. Localization and clustering of recombinant green fluorescent protein-tagged Kv2.1 occurred by the same sequence of events, and imaging of GFP-Kv2.1 clustering in living neurons revealed dynamic fusion events that underlie cluster formation. Overexpression of GFP-Kv2.1 accelerated the clustering program in pyramidal neurons such that the observed differences in Kv2.1 clustering in pyramidal neurons and interneurons were eliminated. Confocal imaging showed a preferential association of Kv2.1 with the basal membrane in cultured neurons, and electrophysiological recordings from neurons cultured on transistors revealed that Kv2.1 contributed the bulk of a previously described adherens junction delayed rectifier potassium conductance. Finally, Kv2.1 clusters were found spatially associated with ryanodine receptor intracellular Ca(2+) ([Ca(2+)](i)) release channels. These findings reveal a stepwise assembly of Kv2.1 potassium channels into membrane clusters during development, and an association of these clusters with Ca(2+) signaling apparatus. Together these data suggest that the restricted localization of Kv2.1 may play an important role in the previously observed contribution of this potassium channel in regulating dendritic [Ca(2+)](i) transients.
Asunto(s)
Dendritas/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Animales , Señalización del Calcio , Células Cultivadas , Canales de Potasio de Tipo Rectificador Tardío , Embrión de Mamíferos , Proteínas Fluorescentes Verdes , Hipocampo/citología , Indicadores y Reactivos , Interneuronas/metabolismo , Proteínas Luminiscentes , Técnicas de Placa-Clamp , Células Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shab , Distribución TisularRESUMEN
The repertoire of Kv1 potassium channels expressed in presynaptic terminals of mammalian central neurons is shaped by intrinsic trafficking signals that determine surface-expression efficiencies of homomeric and heteromeric Kv1 channel complexes. Here, we show that a determinant controlling surface expression of Kv1 channels is localized to the highly conserved pore region. Point-mutation analysis revealed two residues as critical for channel trafficking, one in the extracellular "turret" domain and one in the region distal to the selectivity filter. Interestingly, these same residues also form the binding sites for polypeptide neurotoxins. Our findings demonstrate a previously uncharacterized function for the channel-pore domain as a regulator of channel trafficking.
Asunto(s)
Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Retículo Endoplásmico/metabolismo , Hipocampo/citología , Canal de Potasio Kv.1.1 , Canal de Potasio Kv1.4 , Datos de Secuencia Molecular , Canales de Potasio/genética , RatasRESUMEN
Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K(+) (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 alpha subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A delta and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na(+) channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 alpha subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.
Asunto(s)
Neuronas Aferentes/fisiología , Dolor/fisiopatología , Canales de Potasio/fisiología , Animales , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiología , Masculino , Neuronas Aferentes/metabolismo , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismoRESUMEN
Episodic ataxia type 1 (EA-1) is a neurological disorder arising from mutations in the Kv1.1 potassium channel alpha-subunit. EA-1 patients exhibit substantial phenotypic variability resulting from at least 14 distinct EA-1 point mutations. We found that EA-1 missense mutations generate mutant Kv1.1 subunits with folding and intracellular trafficking properties indistinguishable from wild-type Kv1.1. However, the single identified EA-1 nonsense mutation exhibits intracellular aggregation and detergent insolubility. This phenotype can be transferred to co-assembled Kv1 alpha- and Kv beta-subunits associated with Kv1.1 in neurons. These results suggest that as in many neurodegenerative disorders, intracellular aggregation of misfolded Kv1.1-containing channels may contribute to the pathophysiology of EA-1.
Asunto(s)
Ataxia/genética , Mutación , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Canales de Potasio/metabolismo , Transporte de Proteínas/fisiología , Animales , Ataxia/fisiopatología , Células COS , Humanos , Canal de Potasio Kv.1.1 , Fenotipo , Canales de Potasio/química , Pliegue de Proteína , Ratas , Ubiquitina/metabolismo , Vimentina/metabolismoRESUMEN
For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.
Asunto(s)
Acetatos/metabolismo , Dióxido de Carbono/metabolismo , Ejercicio Físico/fisiología , Glucosa/metabolismo , Consumo de Oxígeno , Resistencia Física/fisiología , Esfuerzo Físico/fisiología , Descanso/fisiología , Bicarbonato de Sodio/metabolismo , Acetatos/administración & dosificación , Adulto , Isótopos de Carbono , Deuterio , Ayuno , Glucosa/administración & dosificación , Humanos , Infusiones Intravenosas , Masculino , Oxidación-Reducción , Periodo Posprandial , Respiración , Bicarbonato de Sodio/administración & dosificaciónRESUMEN
In the mammalian hippocampal formation, dendrotoxin-sensitive voltage-gated K(+) (Kv) channels modulate action potential propagation and neurotransmitter release. To explore the neuroanatomical basis for this modulation, we used in situ hybridization, coimmunoprecipitation, and immunohistochemistry to determine the subcellular localization of the Kv channel subunits Kv1.1, Kv1.2, Kv1.4, and Kvbeta2 within the adult rat hippocampus. Although mRNAs encoding all four of these Kv channel subunits are expressed in the cells of origin of each major hippocampal afferent and intrinsic pathway, immunohistochemical staining suggests that the encoded subunits are associated with the axons and terminal fields of these cells. Using an excitotoxin lesion strategy, we explored the subcellular localization of these subunits in detail. We found that ibotenic acid lesions of the entorhinal cortex eliminated Kv1.1 and Kv1.4 immunoreactivity and dramatically reduced Kv1.2 and Kvbeta2 immunoreactivity in the middle third of the dentate molecular layer, indicating that these subunits are located on axons and terminals of entorhinal afferents. Similarly, ibotenic acid lesions of the dentate gyrus eliminated Kv1.1 and Kv1.4 immunoreactivity in the stratum lucidum of CA3, indicating that these subunits are located on mossy fiber axons. Kainic acid lesions of CA3 dramatically reduced Kv1.1 immunoreactivity in the stratum radiatum of CA1-CA3, indicating that Kv1.1 immunoreactivity in these subfields is associated with the axons and terminals of the Schaffer collaterals. Together with the results of coimmunoprecipitation analyses, these data suggest that action potential propagation and glutamate release at excitatory hippocampal synapses are directly modulated by Kv1 channel complexes predominantly localized on axons and nerve terminals.
Asunto(s)
Hipocampo/metabolismo , Canales de Potasio/metabolismo , Subunidades de Proteína , Animales , Axones/metabolismo , Venenos Elapídicos/farmacología , Fórnix/fisiología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Ácido Iboténico/farmacología , Inmunohistoquímica , Hibridación in Situ , Ácido Kaínico/farmacología , Fibras Musgosas del Hipocampo/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especificidad de Órganos/efectos de los fármacos , Canales de Potasio/análisis , Canales de Potasio/genética , Pruebas de Precipitina , Terminales Presinápticos/metabolismo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , RatasRESUMEN
Voltage-gated Na(+) and K(+) channels are localized to distinct subcellular domains in mammalian myelinated nerve fibers. Specifically, Na(+) channels are clustered in high densities at nodes of Ranvier, while K(+) channels are found in juxtaparanodal zones just beyond regions of axoglial contact where sequential layers of the myelin sheath terminate. Specific targeting, clustering, and maintenance of these channels in their respective domains are essential to achieve high conduction velocities of action potential propagation. The cellular, molecular, and developmental mechanisms that exist to achieve this neuronal specialization are discussed and reviewed. Current evidence points to a prominent role in channel clustering played by myelinating glial cells, and sites of axoglial contact in particular.
Asunto(s)
Canales Iónicos , Neuronas/fisiología , Nódulos de Ranvier/fisiología , Animales , Ratones , Neuronas/química , Canales de Potasio/química , Estructura Terciaria de Proteína , Nódulos de Ranvier/química , Ratas , Transducción de Señal , Canales de Sodio/químicaRESUMEN
Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.
Asunto(s)
Axones/metabolismo , Vaina de Mielina/metabolismo , Nervio Óptico/crecimiento & desarrollo , Canales de Sodio/metabolismo , Animales , Axones/ultraestructura , Inmunohistoquímica , Ratones , Ratones Mutantes Neurológicos/anatomía & histología , Ratones Mutantes Neurológicos/crecimiento & desarrollo , Ratones Mutantes Neurológicos/metabolismo , Vaina de Mielina/ultraestructura , Nervio Óptico/metabolismo , Nervio Óptico/ultraestructura , Nervios Periféricos/crecimiento & desarrollo , Nervios Periféricos/metabolismo , Nervios Periféricos/ultraestructura , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Nódulos de Ranvier/metabolismo , Nódulos de Ranvier/ultraestructura , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/ultraestructura , Canales de Sodio/genéticaRESUMEN
Related to hepatic autoregulation we evaluated hypotheses that 1) glucose production would be altered as a result of a glycerol load, 2) decreased glucose recycling rate (Rr) would result from increased glycerol uptake, and 3) the absolute rate of gluconeogenesis (GNG) from glycerol would be positively correlated to glycerol rate of disappearance (R(d)) during a glycerol load. For these purposes, glucose and glycerol kinetics were determined in eight men during rest and during 90 min of leg cycle ergometry at 45 and 65% of peak O2 consumption (.VO2 (peak)). Trials were conducted after an overnight fast, with exercise commencing 12 h after the last meal. Subjects received a continuous infusion of [6,6-(2)H(2)]glucose, [1-(13)C]glucose, and [1,1,2,3,3-(2)H(5)]glycerol without (CON) or with an additional 1,000 mg (rest: 20 mg/min; exercise: 40 mg/min) of [2-(13)C]- or unlabeled glycerol added to the infusate (GLY). Infusion of glycerol dampened glucose Rr, calculated as the difference between [6,6-(2)H(2)]- and [1-(13)C]glucose rates of appearance (R(a)), at rest [0.35 +/- 0.12 (CON) vs. 0.12 +/- 0.10 mg. kg(-1). min(-1) (GLY), P < 0.05] and during exercise at both intensities [45%: 0.63 +/- 0.14 (CON) vs. 0.04 +/- 0.12 (GLY); 65%: 0.73 +/- 0.14 (CON) vs. 0.04 +/- 0.17 mg. kg(-1). min(-1) (GLY), P < 0.05]. Glucose R(a) and oxidation were not affected by glycerol infusion at rest or during exercise. Throughout rest and both exercise intensities, glycerol R(d) was greater in GLY vs. CON conditions (rest: 0.30 +/- 0.04 vs. 0.58 +/- 0.04; 45%: 0.57 +/- 0.07 vs. 1.19 +/- 0.04; 65%: 0.73 +/- 0.06 vs. 1.27 +/- 0.05 mg. kg(-1). min(-1), CON vs. GLY, respectively). Differences in glycerol R(d) (DeltaR(d)) between protocols equaled the unlabeled glycerol infusion rate and correlated with plasma glycerol concentration (r = 0.97). We conclude that infusion of a glycerol load during rest and exercise at 45 and 65% of .VO2(peak) 1) does not affect glucose R(a) or R(d), 2) blocks glucose Rr, 3) increases whole body glycerol R(d) in a dose-dependent manner, and 4) results in gluconeogenic rates from glycerol equivalent to CON glucose recycling rates.
Asunto(s)
Ejercicio Físico/fisiología , Gluconeogénesis/fisiología , Glicerol/farmacología , Homeostasis , Adulto , Registros de Dieta , Glucosa/metabolismo , Glicerol/metabolismo , Humanos , Cinética , Masculino , Oxidación-Reducción , Intercambio Gaseoso Pulmonar , Descanso/fisiologíaRESUMEN
Functional NMDA receptors are heteromultimeric complexes of the NR1 subunit in combination with at least one of the four NR2 subunits (A-D). Coexpression of NR3A, an additional subunit of the NMDA receptor family, modifies NMDA-mediated responses. It is unclear whether NR3A interacts directly with NR1 and/or NR2 subunits and how such association might regulate the intracellular trafficking and membrane expression of NR3A. Here we show that NR3A coassembles with NR1-1a and NR2A to form a receptor complex with distinct single-channel properties and a reduced relative calcium permeability. NR3A associates independently with both NR1-1a and NR2A in the endoplasmic reticulum, but only heteromeric complexes containing the NR1-1a NMDA receptor subunit are targeted to the plasma membrane. Homomeric NR3A complexes or complexes composed of NR2A and NR3A were not detected on the cell surface and are retained in the endoplasmic reticulum. Coexpression of NR1-1a facilitates the surface expression of NR3A-containing receptors, reduces the accumulation of NR3A subunits in the endoplasmic reticulum, and induces the appearance of intracellular clusters where both subunits are colocalized. Our data demonstrate a role for subunit oligomerization and specifically assembly with the NR1 subunit in the trafficking and plasma membrane targeting of the receptor complex.
Asunto(s)
Riñón/metabolismo , Subunidades de Proteína , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Líquido Intracelular/metabolismo , Riñón/citología , Técnicas de Placa-Clamp , Permeabilidad , Pruebas de Precipitina , Ratas , Receptores de N-Metil-D-Aspartato/genética , TransfecciónRESUMEN
Axonal K+ channels involved in normal spinal cord function are candidate targets for therapeutics, which improve sensorimotor function in spinal cord injury. To this end, we have investigated the expression, localization, and coassociation of Kv1 alpha and beta subunits in human, rat, and bovine spinal cord. We find that Kv1.1, Kv1.2, and Kvbeta2 form heteromultimeric complexes at juxtaparanodal zones in myelinated fibers. However, these same complexes are also present in paranodal regions of some spinal cord axons, and staining with antibodies against Caspr, a component of the paranodal axoglial junction, overlaps with these paranodal K+ channels. This latter observation suggests a unique role for these channels in normal spinal cord function and may provide an explanation for the sensitivity of spinal cord to K+ channel blockers. Moreover, the conservation of these characteristics between human, rat, and bovine nodes of Ranvier suggests an essential role for this defined channel complex in spinal cord function.
Asunto(s)
Fibras Nerviosas Mielínicas/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Nódulos de Ranvier/metabolismo , Médula Espinal/metabolismo , Animales , Bovinos , Humanos , Canal de Potasio Kv.1.1 , Canal de Potasio Kv.1.2 , Fibras Nerviosas Mielínicas/ultraestructura , Canales de Potasio/química , Nódulos de Ranvier/ultraestructura , Ratas , Médula Espinal/citologíaRESUMEN
Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.
Asunto(s)
Epitelio/metabolismo , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Citoplasma/metabolismo , Perros , Endocitosis , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Receptores de Glutamato Metabotrópico/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Shaker-related or Kv1 voltage-gated K(+) channels play critical roles in regulating the excitability of mammalian neurons. Native Kv1 channel complexes are octamers of four integral membrane alpha subunits and four cytoplasmic beta subunits, such that a tremendous diversity of channel complexes can be assembled from the array of alpha and beta subunits expressed in the brain. However, biochemical and immunohistochemical studies have demonstrated that only certain complexes predominate in the mammalian brain, suggesting that regulatory mechanisms exist that ensure plasma membrane targeting of only physiologically appropriate channel complexes. Here we show that Kv1 channels assembled as homo- or heterotetrameric complexes had distinct surface expression characteristics in both transfected mammalian cells and hippocampal neurons. Homotetrameric Kv1.1 channels were localized to endoplasmic reticulum, Kv1.4 channels to the cell surface, and Kv1.2 channels to both endoplasmic reticulum and the cell surface. Heteromeric assembly with Kv1.4 resulted in dose-dependent increases in cell surface expression of coassembled Kv1.1 and Kv1.2, while coassembly with Kv1.1 had a dominant-negative effect on Kv1.2 and Kv1.4 surface expression. Coassembly with Kv beta subunits promoted cell surface expression of each Kv1 heteromeric complex. These data suggest that subunit composition and stoichiometry determine surface expression characteristics of Kv1 channels in excitable cells.
Asunto(s)
Canales de Potasio/análisis , Animales , Células COS , Membrana Celular/metabolismo , Hipocampo/metabolismo , Canales de Potasio/biosíntesis , Canales de Potasio/genética , Conformación Proteica , TransfecciónRESUMEN
The discrete localization of ion channels is a critical determinant of neuronal excitability. We show here that the dendritic K+ channels Kv2.1 and Kv2.2 were differentially targeted in cultured hippocampal neurons. Kv2.1 was found in high-density clusters on the soma and proximal dendrites, while Kv2.2 was uniformly distributed throughout the soma and dendrites. Chimeras revealed a proximal restriction and clustering domain on the cytoplasmic tail of Kv2.1. Truncations and internal deletions revealed a 26-amino acid targeting signal within which four residues were critical for localization. This signal is not related to other known sequences for neuronal and epithelial membrane protein targeting and represents a novel cytoplasmic signal responsible for proximal restriction and clustering.