RESUMEN
New neurotechnologies fueled by the BRAIN Initiative now allow investigators to map, monitor and modulate complex neural circuits, enabling the pursuit of research questions previously considered unapproachable. Yet it is the convergence of molecular neuroscience with the new systems neuroscience that promises the greatest future advances. This is particularly true for our understanding of nervous system disorders, some of which have known molecular drivers or pathology but result in unknown perturbations in circuit function. NIH-supported research on "BRAIN Circuits" programs integrate experimental, analytic, and theoretical capabilities for analysis of specific neural circuits and their contributions to perceptions, motivations, and actions. In this review, we describe the BRAIN priority areas, review our strategy for balancing early feasibility with mature projects, and the balance of individual with team science for this 'BRAIN Circuits' program. We also highlight the diverse portfolio of techniques, species, and neural systems represented in these projects.
Asunto(s)
Encéfalo , Neurociencias , Mapeo Encefálico , Sistema Nervioso CentralAsunto(s)
Bases de Datos Factuales , National Institutes of Health (U.S.)/economía , National Institutes of Health (U.S.)/estadística & datos numéricos , Apoyo a la Investigación como Asunto/economía , Apoyo a la Investigación como Asunto/estadística & datos numéricos , Algoritmos , Inteligencia Artificial , Análisis por Conglomerados , Gráficos por Computador , Minería de Datos , Estados Unidos , Interfaz Usuario-ComputadorRESUMEN
General anesthetics cause sedation, hypnosis, and immobilization via CNS mechanisms that remain incompletely understood; contributions of particular anesthetic targets in specific neural pathways remain largely unexplored. Among potential molecular targets for mediating anesthetic actions, members of the TASK subgroup [TASK-1 (K2P3.1) and TASK-3 (K2P9.1)] of background K(+) channels are appealing candidates since they are expressed in CNS sites relevant to anesthetic actions and activated by clinically relevant concentrations of inhaled anesthetics. Here, we used global and conditional TASK channel single and double subunit knock-out mice to demonstrate definitively that TASK channels account for motoneuronal, anesthetic-activated K(+) currents and to test their contributions to sedative, hypnotic, and immobilizing anesthetic actions. In motoneurons from all knock-out mice lines, TASK-like currents were reduced and cells were less sensitive to hyperpolarizing effects of halothane and isoflurane. In an immobilization assay, higher concentrations of both halothane and isoflurane were required to render TASK knock-out animals unresponsive to a tail pinch; in assays of sedation (loss of movement) and hypnosis (loss-of-righting reflex), TASK knock-out mice showed a modest decrease in sensitivity, and only for halothane. In conditional knock-out mice, with TASK channel deletion restricted to cholinergic neurons, immobilizing actions of the inhaled anesthetics and sedative effects of halothane were reduced to the same extent as in global knock-out lines. These data indicate that TASK channels in cholinergic neurons are molecular substrates for select actions of inhaled anesthetics; for immobilization, which is spinally mediated, these data implicate motoneurons as the likely neuronal substrates.
Asunto(s)
Anestésicos por Inhalación/farmacología , Pérdida de Tono Postural/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Tronco Encefálico/citología , Línea Celular Transformada , Colina O-Acetiltransferasa/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Femenino , Eliminación de Gen , Halotano/farmacología , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Isoflurano/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Técnicas de Placa-Clamp , Canales de Potasio de Dominio Poro en Tándem/deficienciaRESUMEN
When inappropriate for salt status, the mineralocorticoid aldosterone induces cardiac and renal injury. Autonomous overproduction of aldosterone from the adrenal zona glomerulosa (ZG) is also the most frequent cause of secondary hypertension. Yet, the etiology of nontumorigenic primary hyperaldosteronism caused by bilateral idiopathic hyperaldosteronism remains unknown. Here, we show that genetic deletion of TWIK-related acid-sensitive K (TASK)-1 and TASK-3 channels removes an important background K current that results in a marked depolarization of ZG cell membrane potential. Although TASK channel deletion mice (TASK-/-) adjust urinary Na excretion and aldosterone production to match Na intake, they produce more aldosterone than control mice across the range of Na intake. Overproduction of aldosterone is not the result of enhanced activity of the renin-angiotensin system because circulating renin concentrations remain either unchanged or lower than those of control mice at each level of Na intake. In addition, TASK-/- mice fail to suppress aldosterone production in response to dietary Na loading. Autonomous aldosterone production is also demonstrated by the failure of an angiotensin type 1 receptor blocker, candesartan, to normalize aldosterone production to control levels in TASK-/- mice. Thus, TASK-/- channel knockout mice exhibit the hallmarks of primary hyperaldosteronism. Our studies establish an animal model of nontumorigenic primary hyperaldosteronism and identify TASK channels as a possible therapeutic target for primary hyperaldosteronism.
Asunto(s)
Hiperaldosteronismo/genética , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Aldosterona/orina , Animales , Secuencia de Bases , Cartilla de ADN , Hibridación in Situ , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem/genética , RadioinmunoensayoRESUMEN
Central respiratory chemoreception is the mechanism by which the CNS maintains physiologically appropriate pH and PCO2 via control of breathing. A prominent hypothesis holds that neural substrates for this process are distributed widely in the respiratory network, especially because many neurons that make up this network are chemosensitive in vitro. We and others have proposed that TASK channels (TASK-1, K(2P)3.1 and/or TASK-3, K(2P)9.1) may serve as molecular sensors for central chemoreception because they are highly expressed in multiple neuronal populations in the respiratory pathway and contribute to their pH sensitivity in vitro. To test this hypothesis, we examined the chemosensitivity of two prime candidate chemoreceptor neurons in vitro and tested ventilatory responses to CO2 using TASK channel knock-out mice. The pH sensitivity of serotonergic raphe neurons was abolished in TASK channel knock-outs. In contrast, pH sensitivity of neurons in the mouse retrotrapezoid nucleus (RTN) was fully maintained in a TASK null background, and pharmacological evidence indicated that a K+ channel with properties distinct from TASK channels contributes to the pH sensitivity of rat RTN neurons. Furthermore, the ventilatory response to CO2 was completely retained in single or double TASK knock-out mice. These data rule out a strict requirement for TASK channels or raphe neurons in central respiratory chemosensation. Furthermore, they indicate that a non-TASK K+ current contributes to chemosensitivity of RTN neurons, which are profoundly pH-sensitive and capable of driving respiratory output in response to local pH changes in vivo.
Asunto(s)
Células Quimiorreceptoras/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Canales de Potasio/fisiología , Centro Respiratorio/fisiología , Animales , Células Cultivadas , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Canales de Potasio de Dominio Poro en Tándem/genética , Núcleos del Rafe/citología , Núcleos del Rafe/metabolismo , Núcleos del Rafe/fisiología , Ratas , Ratas Sprague-Dawley , Centro Respiratorio/citología , Centro Respiratorio/metabolismoRESUMEN
Two-pore-domain K(+) channels provide neuronal background currents that establish resting membrane potential and input resistance; their modulation provides a prevalent mechanism for regulating cellular excitability. The so-called TASK channel subunits (TASK-1 and TASK-3) are widely expressed, and they are robustly inhibited by receptors that signal through Galphaq family proteins. Here, we manipulated G protein expression and membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels in intact and cell-free systems to provide electrophysiological and biochemical evidence that inhibition of TASK channels by Galphaq-linked receptors proceeds unabated in the absence of phospholipase C (PLC) activity, and instead involves association of activated Galphaq subunits with the channels. Receptor-mediated inhibition of TASK channels was faster and less sensitive to a PLCbeta1-ct minigene construct than inhibition of PIP(2)-sensitive Kir3.4(S143T) homomeric channels that is known to be dependent on PLC. TASK channels were strongly inhibited by constitutively active Galphaq, even by a mutated version that is deficient in PLC activation. Receptor-mediated TASK channel inhibition required exogenous Galphaq expression in fibroblasts derived from Galphaq/11 knockout mice, but proceeded unabated in a cell line in which PIP(2) levels were reduced by regulated overexpression of a lipid phosphatase. Direct application of activated Galphaq, but not other G protein subunits, inhibited TASK channels in excised patches, and constitutively active Galphaq subunits were selectively coimmunoprecipitated with TASK channels. These data indicate that receptor-mediated TASK channel inhibition is independent of PIP(2) depletion, and they suggest a mechanism whereby channel modulation by Galphaq occurs through direct interaction with the ion channel or a closely associated intermediary.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Línea Celular , Sistema Libre de Células , Electrofisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Humanos , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Spodoptera , Fosfolipasas de Tipo C/metabolismoRESUMEN
General anesthetics have been a mainstay of surgical practice for more than 150 years, but the mechanisms by which they mediate their important clinical actions remain unclear. Ion channels represent important anesthetic targets, and, although GABA(A) receptors have emerged as major contributors to sedative, immobilizing, and hypnotic effects of intravenous anesthetics, a role for those receptors is less certain in the case of inhalational anesthetics. The neuronal hyperpolarization-activated pacemaker current (Ih) is essential for oscillatory and integrative properties in numerous cell types. Here, we show that clinically relevant concentrations of inhalational anesthetics modulate neuronal Ih and the corresponding HCN channels in a subunit-specific and cAMP-dependent manner. Anesthetic inhibition of Ih involves a hyperpolarizing shift in voltage dependence of activation and a decrease in maximal current amplitude; these effects can be ascribed to HCN1 and HCN2 subunits, respectively, and both actions are recapitulated in heteromeric HCN1-HCN2 channels. Mutagenesis and simulations suggest that apparently distinct actions of anesthetics on V(1/2) and amplitude represent different manifestations of a single underlying mechanism (i.e., stabilization of channel closed state), with the predominant action determined by basal inhibition imposed by individual subunit C-terminal domains and relieved by cAMP. These data reveal a molecular basis for multiple actions of anesthetics on neuronal HCN channels, highlight the importance of proximal C terminus in modulation of HCN channel gating by diverse agents, and advance neuronal pacemaker channels as potentially relevant targets for clinical actions of inhaled anesthetics.
Asunto(s)
Anestésicos/farmacología , Encéfalo/fisiología , Canales Iónicos/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Secuencia de Bases , Tronco Encefálico/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Cartilla de ADN , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Hibridación in Situ , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales Iónicos/genética , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio , Ratas , Mapeo Restrictivo , Tálamo/fisiologíaRESUMEN
It has long been suspected that sensory signal transmission is inhibited in the mammalian brain during sleep. We hypothesized that Cav3.1 T-type Ca2+ channel currents inhibit thalamic sensory transmission to promote sleep. We found that T-type Ca2+ channel activation caused prolonged inhibition (>9 s) of action-potential firing in thalamic projection neurons of WT but not Cav3.1 knockout mice. Inhibition occurred with synaptic transmission blocked and required an increase of intracellular Ca2+. Furthermore, focal deletion of the gene encoding Cav3.1 from the rostral-midline thalamus by using Cre/loxP recombination led to frequent and prolonged arousal, which fragmented and reduced sleep. Interestingly, sleep was not disturbed when Cav3.1 was deleted from cortical pyramidal neurons. These findings support the hypothesis that thalamic T-type Ca2+ channels are required to block transmission of arousal signals through the thalamus and to stabilize sleep.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Sueño/fisiología , Tálamo/fisiología , Potenciales de Acción/fisiología , Animales , Calcio/fisiología , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/genética , Hibridación in Situ , Técnicas In Vitro , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligodesoxirribonucleótidos Antisentido , Técnicas de Placa-ClampRESUMEN
Background potassium currents carried by the KCNK family of two-pore-domain K+ channels are important determinants of resting membrane potential and cellular excitability. TWIK-related acid-sensitive K+ 1 (TASK-1, KCNK3) and TASK-3 (KCNK9) are pH-sensitive subunits of the KCNK family that are closely related and coexpressed in many brain regions. There is accumulating evidence that these two subunits can form heterodimeric channels, but this evidence remains controversial. In addition, a substantial contribution of heterodimeric TASK channels to native currents has not been unequivocally established. In a heterologous expression system, we verified formation of heterodimeric TASK channels and characterized their properties; TASK-1 and TASK-3 were coimmunoprecipitated from membranes of mammalian cells transfected with the channel subunits, and a dominant negative TASK-1(Y191F) construct strongly diminished TASK-3 currents. Tandem-linked heterodimeric TASK channel constructs displayed a pH sensitivity (pK approximately 7.3) in the physiological range closer to that of TASK-1 (pK approximately 7.5) than TASK-3 (pK approximately 6.8). On the other hand, heteromeric TASK channels were like TASK-3 insofar as they were activated by high concentrations of isoflurane (0.8 mm), whereas TASK-1 channels were inhibited. The pH and isoflurane sensitivities of native TASK-like currents in hypoglossal motoneurons, which strongly express TASK-1 and TASK-3 mRNA, were best represented by TASK heterodimeric channels. Moreover, after blocking homomeric TASK-3 channels with ruthenium red, we found a major component of motoneuronal isoflurane-sensitive TASK-like current that could be attributed to heteromeric TASK channels. Together, these data indicate that TASK-1 and TASK-3 subunits coassociate in functional channels, and heteromeric TASK channels provide a substantial component of background K(+) current in motoneurons with distinct modulatory properties.
Asunto(s)
Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Dimerización , Proteínas de Drosophila , Humanos , Concentración de Iones de Hidrógeno , Nervio Hipogloso/citología , Nervio Hipogloso/efectos de los fármacos , Nervio Hipogloso/metabolismo , Transporte Iónico/efectos de los fármacos , Isoflurano/farmacología , Riñón , Datos de Secuencia Molecular , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/química , Multimerización de Proteína , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiología , Rojo de Rutenio/farmacología , TransfecciónRESUMEN
TASK-1 and TASK-3 are functional members of the tandem-pore K+ (K2P) channel family, and mRNAs for both channels are expressed together in many brain regions. Although TASK-1 and TASK-3 subunits are able to form heteromers when their complementary RNAs are injected into oocytes, whether functional heteromers are present in the native tissue is not known. Using cultured cerebellar granule (CG) neurones that express mRNAs of both TASK-1 and TASK-3, we studied the presence of heteromers by comparing the sensitivities of cloned and native K+ channels to extracellular pH (pHo) and ruthenium red. The single-channel conductance of TASK-1, TASK-3 and a tandem construct (TASK-1/TASK-3) expressed in COS-7 cells were 14.2 +/- 0.4, 37.8 +/- 0.7 and 38.1 +/- 0.7 pS (-60 mV), respectively. TASK-3 and TASK-1/TASK-3 (and TASK-3/TASK-1) displayed nearly identical single-channel kinetics. TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 26 +/- 4 and 36 +/- 2 %, respectively, when pHo was changed from 8.3 to 7.3. In outside-out patches from CG neurones, the K+ channel with single channel properties similar to those of TASK-3 was inhibited by 31 +/- 7 % by the same reduction in pHo. TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 78 +/- 7 and 3 +/- 4 %, respectively, when 5 microm ruthenium red was applied to outside-out patches. In outside-out patches from CG neurones containing a 38 pS channel, two types of responses to ruthenium red were observed. Ruthenium red inhibited the channel activity by 77 +/- 5 % in 42 % of patches (range: 72-82 %) and by 5 +/- 4 % (range: 0-9 %) in 58 % of patches. When patches contained more than three 38 pS channels, the average response to ruthenium red was 47 +/- 6 % inhibition (n= 5). These electrophysiological studies show that native 38 pS K+ channels of the TASK family in cultured CG neurones consist of both homomeric TASK-3 and heteromeric TASK-1/TASK-3.
Asunto(s)
Cerebelo/fisiología , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/genética , Animales , Células COS , Cerebelo/citología , Expresión Génica , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas del Tejido Nervioso/metabolismo , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Ratas , Rojo de Rutenio/farmacología , TransfecciónRESUMEN
Occulocutaneous albinism is caused by mutations in the gene encoding the enzyme tyrosinase. Individuals with this disorder are predisposed to visual system deficits. We determined the critical period during development when tyrosinase expression is essential for the appropriate pathfinding of ganglion cell axons from the retina to the dorsal lateral geniculate nucleus. We used a line of mice with a Tyrosinase transgene, the expression of which is regulatable with the lac operator-repressor system, to restrict tyrosinase activity to discrete periods of embryogenesis. When tyrosinase was expressed throughout the period of neuroblast divisions that produce the ipsilaterally projecting ganglion cells, axonal projections innervated the same volume of the ipsilateral dorsal lateral geniculate nucleus of the thalamus as in normal mice. If tyrosinase expression ceased before the end of neuroblast divisions, or was not initiated until after they had begun, the degree of ipsilateral innervation was smaller, as in albino mice. Tyrosinase expression was not required during the entire period of pathfinding itself or during final maturation of the retinogeniculate pathway. Thus, tyrosinase appears to set up a signal early in visual system development that determines the pathway taken later by ganglion cell axons.
Asunto(s)
Cuerpos Geniculados/embriología , Monofenol Monooxigenasa/metabolismo , Retina/embriología , Células Ganglionares de la Retina/enzimología , Vías Visuales/embriología , Vías Visuales/enzimología , Animales , Axones/ultraestructura , Proteínas Bacterianas/genética , División Celular , Cuerpos Geniculados/citología , Cinética , Represoras Lac , Ratones , Ratones Transgénicos , Monofenol Monooxigenasa/genética , Movimiento , Proteínas Represoras/genética , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Vías Visuales/citologíaRESUMEN
Neuronal G protein-coupled inwardly-rectifying potassium channels (GIRKs, Kir3.x) can be activated or inhibited by distinct classes of receptors (Galphai/o and Galphaq/11-coupled, respectively), providing dynamic regulation of neuronal excitability. In this mini-review, we highlight findings from our laboratory in which we used a mammalian heterologous expression system to address mechanisms of GIRK channel regulation by Galpha and Gbetagamma subunits. We found that, like beta1- and beta2-containing Gbetagamma dimers, GIRK channels are also activated by G protein betagamma dimers containing beta3 and beta4 subunits. By contrast, GIRK currents are inhibited by beta5-containing Gbetagamma dimers and/or by Galpha proteins of the Galphaq/11 family. The properties of Gbeta5-mediated inhibition suggest that beta5-containing Gbetagamma dimers act as competitive antagonists of other activating Gbetagamma pairs on GIRK channels. Inhibition of GIRK channels by Galpha subunits is specific to members of the Galphaq/11 family and appears to result, at least in part, from activation of phospholipase C (PLC) and the resultant decrease in membrane levels of phosphatidylinositol-4,5-bisphosphate (PIP2), an endogenous co-factor necessary for GIRK channel activity; this Galphaq/11 activated mechanism is largely responsible for receptor-mediated GIRK channel inhibition.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Unión Competitiva , Línea Celular , Dimerización , Activación Enzimática , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Activación del Canal Iónico , Transporte Iónico , Riñón , Potenciales de la Membrana , Fosfatidilinositol 4,5-Difosfato/fisiología , Fosfatidilinositol Diacilglicerol-Liasa , Potasio/metabolismo , Canales de Potasio/química , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/fisiología , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
Leak K+ currents contribute to the resting membrane potential and are important for modulation of neuronal excitability. Within the past few years, an entire family of genes has been described whose members form leak K+ channels, insofar as they generate potassium-selective currents with little voltage- and time-dependence. They are often referred to as "two-pore-domain" channels because of their predicted topology, which includes two pore-forming regions in each subunit. These channels are modulated by a host of different endogenous and clinical compounds such as neurotransmitters and anesthetics, and by physicochemical factors such as temperature, pH, oxygen tension, and osmolarity. They also are subject to long-term regulation by changes in gene expression. In this review, the authors describe multiple roles that modulation of leak K+ channels play in CNS function and discuss evidence that members of the two-pore-domain family are molecular substrates for these processes.
Asunto(s)
Sistema Nervioso Central/fisiología , Neuronas/fisiología , Canales de Potasio/fisiología , Anestésicos/farmacología , Animales , Encéfalo/fisiología , Sistema Nervioso Central/metabolismo , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Potenciales de la Membrana , Neuronas/metabolismo , Neurotransmisores/fisiología , Concentración Osmolar , Oxígeno/fisiología , Canales de Potasio/metabolismo , ARN/metabolismo , TemperaturaAsunto(s)
Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/metabolismo , Anestésicos por Inhalación/metabolismo , Animales , Clonación Molecular , Electrofisiología , Familia de Multigenes , Proteínas del Tejido Nervioso , Neuronas/citología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Fenómenos Fisiológicos RespiratoriosRESUMEN
TASK-1 and TASK-3, members of the two-pore-domain channel family, are widely expressed leak potassium channels responsible for maintenance of cell membrane potential and input resistance. They are sites of action for a variety of modulatory agents, including volatile anesthetics and neurotransmitters/hormones, the latter acting via mechanisms that have remained elusive. To clarify these mechanisms, we generated mutant channels and found that alterations disrupting anesthetic (halothane) activation of these channels also disrupted transmitter (thyrotropin-releasing hormone, TRH) inhibition and did so to a similar degree. For both TASK-1 and TASK-3, mutations (substitutions with corresponding residues from TREK-1) in a six-residue sequence at the beginning of the cytoplasmic C terminus virtually abolished both anesthetic activation and transmitter inhibition. The only sequence motif identified with a classical signaling mechanism in this region is a potential phosphorylation site; however, mutation of this site failed to disrupt modulation. TASK-1 and TASK-3 differed insofar as a large portion of the C terminus was necessary for the full effects of halothane and TRH on TASK-3 but not on TASK-1. Finally, tandem-linked TASK-1/TASK-3 heterodimeric channels were fully modulated by anesthetic and transmitter, and introduction of the identified mutations either into the TASK-1 or the TASK-3 portion of the channel was sufficient to disrupt both effects. Thus, both anesthetic activation and transmitter inhibition of these channels require a region at the interface between the final transmembrane domain and the cytoplasmic C terminus that has not been associated previously with receptor signal transduction. Our results also indicate a close molecular relationship between these two forms of modulation, one endogenous and the other clinically applied.
Asunto(s)
Anestésicos por Inhalación/farmacología , Proteínas del Tejido Nervioso , Neurotransmisores/farmacología , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/metabolismo , Animales , Línea Celular , Halotano/farmacología , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación Puntual , Canales de Potasio/genética , Ratas , Relación Estructura-Actividad , Hormona Liberadora de Tirotropina/farmacologíaRESUMEN
The recently described two-pore-domain K+ channels, TASK-1 and TASK-3, generate currents with a unique set of properties; specifically, the channels produce instantaneous open-rectifier (i.e., "leak") K+ currents that are modulated by extracellular pH and by clinically useful anesthetics. In this study, we used histochemical and in vitro electrophysiological approaches to determine that TASK channels are expressed in serotonergic raphe neurons and to show that they confer a pH and anesthetic sensitivity to these neurons. By combining in situ hybridization for TASK-1 or TASK-3 with immunohistochemical localization of tryptophan hydroxylase, we found that a majority of serotonergic neurons in both dorsal and caudal raphe cell groups contain TASK channel transcripts (approximately 70-90%). Whole-cell voltage-clamp recordings were obtained from raphe cells that responded to 5-HT in a manner characteristic of serotonergic neurons (i.e., with activation of an inwardly rectifying K+ current). In those cells, we isolated an endogenous K+ conductance that had properties expected of TASK channel currents; raphe neurons expressed a joint pH- and halothane-sensitive open-rectifier K+ current. The pH sensitivity of this current (pK approximately 7.0) was intermediate between that of TASK-1 and TASK-3, consistent with functional expression of both channel types. Together, these data indicate that TASK-1 and TASK-3 are expressed and functional in serotonergic raphe neurons. The pH-dependent inhibition of TASK channels in raphe neurons may contribute to ventilatory and arousal reflexes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by volatile anesthetics could play a role in their immobilizing and sedative-hypnotic effects.