RESUMEN
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Rodopsinas Microbianas/metabolismo , Cationes Monovalentes/metabolismo , Canales de Cloruro/aislamiento & purificación , Canales de Cloruro/efectos de la radiación , Canales de Cloruro/ultraestructura , Cristalografía/métodos , Radiación Electromagnética , Rayos Láser , Simulación de Dinámica Molecular , Nocardioides , Conformación Proteica en Hélice alfa/efectos de la radiación , Estructura Terciaria de Proteína/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Proteínas Recombinantes/ultraestructura , Retinaldehído/metabolismo , Retinaldehído/efectos de la radiación , Rodopsinas Microbianas/aislamiento & purificación , Rodopsinas Microbianas/efectos de la radiación , Rodopsinas Microbianas/ultraestructura , Agua/metabolismoRESUMEN
Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl- dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl- in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl- concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl- non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl- conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl-.
Asunto(s)
Channelrhodopsins/química , Canales de Cloruro/química , Criptófitas/metabolismo , Channelrhodopsins/metabolismo , Channelrhodopsins/efectos de la radiación , Canales de Cloruro/metabolismo , Canales de Cloruro/efectos de la radiación , Cloruros/metabolismo , Activación del Canal Iónico , Luz , Optogenética/métodosRESUMEN
In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.
Asunto(s)
Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/fisiología , Área Preóptica/citología , Área Preóptica/fisiología , Sueño/fisiología , Transcriptoma , Animales , Biomarcadores/análisis , Channelrhodopsins , Canales de Cloruro/metabolismo , Canales de Cloruro/efectos de la radiación , Colecistoquinina/análisis , Colecistoquinina/genética , Hormona Liberadora de Corticotropina/análisis , Hormona Liberadora de Corticotropina/genética , Femenino , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/efectos de la radiación , Área Hipotalámica Lateral/fisiología , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Optogenética , Área Preóptica/efectos de los fármacos , Área Preóptica/efectos de la radiación , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Sueño/efectos de los fármacos , Sueño/efectos de la radiación , Taquicininas/análisis , Taquicininas/genética , Vigilia/fisiología , Vigilia/efectos de la radiaciónRESUMEN
BACKGROUND: Excessive skin exposure to solar radiation damages proteins and DNA, ultimately leading to skin ageing and cancers. OBJECTIVES: To identify new ultraviolet B (UVB) target genes to understand the mechanisms behind the detrimental effects of UVB. METHODS: Organotypic, stratified cultures of rat keratinocytes were exposed to UVB and analysed using a genome-wide expression array, quantitative real-time polymerase chain reaction and histology. The most downregulated gene, rClca2, was further characterized in rat keratinocytes and mouse skin models. RESULTS: A single, 30 mJ cm(-2) dose of broadband UVB proved effective in the organotypic epidermal culture. The expression of 627 genes was changed 24 h postirradiation. In silico analysis of the data indicated activation of DNA repair, metabolism, cell cycle control and amino acid metabolism, but only limited inflammation under these conditions. We selected for further investigation the most downregulated gene, rClca2, previously suggested to regulate keratinocyte differentiation and adhesion, and found that UVB caused a long-lasting downregulation in its expression. Both the rClca2 full-length isoform (expressed in the differentiating cells) and the truncated isoform (expressed in the basal layers) were reduced by UVB. Immunohistochemistry of mouse skin samples with isoform-specific antibodies showed a similar, epidermal differentiation-related pattern. In mouse specimens exposed to chronic ultraviolet radiation (UVR) the staining intensities were reduced and the differentiation-related isoform was disturbed in the hyperplastic and carcinomatous areas induced by UVR. CONCLUSIONS: The data show that rClca2 is a novel UVB target gene and suggest that it might play a role in epidermal differentiation and UV-dependent skin malignancies.
Asunto(s)
Canales de Cloruro/efectos de la radiación , Epidermis/efectos de la radiación , Rayos Ultravioleta , Animales , Diferenciación Celular/efectos de la radiación , Células Cultivadas , Canales de Cloruro/metabolismo , Relación Dosis-Respuesta en la Radiación , Regulación hacia Abajo , Células Epidérmicas , Epidermis/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Queratinocitos/efectos de la radiación , Ratones , ARN/metabolismo , Ratas , Factores de Transcripción/efectos de la radiaciónRESUMEN
Five hundred and thirty-two nanometers laser light evokes neuron-specific electrical responses in identified neurons of Helix ganglia. Such responses are intensity-dependent over the range 25-1500 mW, readily reversible and repeatable. Detailed experiments on the C1 neuron, which is inhibited by 532 nm light, showed that inhibition results from a selective increase in transmembrane Cl(-) ion conductance. Experiments with calcium-sensitive microelectrodes suggest that the response does not result from an increase in [Ca(2+)](i). The change in Cl(-) ion conductance probably occurs in the extensive plasmalemma infoldings of the proximal axon.
Asunto(s)
Membrana Celular/efectos de la radiación , Canales de Cloruro/efectos de la radiación , Caracoles Helix/efectos de la radiación , Luz , Sistema Nervioso/efectos de la radiación , Neuronas/efectos de la radiación , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Axones/metabolismo , Axones/efectos de la radiación , Membrana Celular/metabolismo , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/metabolismo , Ganglios de Invertebrados/efectos de la radiación , Caracoles Helix/citología , Caracoles Helix/metabolismo , Rayos Láser , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Inhibición Neural/fisiología , Inhibición Neural/efectos de la radiación , Neuronas/citología , Neuronas/metabolismo , Estimulación Luminosa , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de la radiaciónRESUMEN
Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.
Asunto(s)
Calcio/farmacocinética , Calcio/efectos de la radiación , Inositol 1,4,5-Trifosfato/análogos & derivados , Fotólisis/efectos de la radiación , Glándula Submandibular/citología , Glándula Submandibular/metabolismo , Animales , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/efectos de la radiación , Relación Dosis-Respuesta a Droga , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/efectos de la radiación , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/efectos de la radiación , Ratas , Ratas Wistar , Glándula Submandibular/efectos de la radiaciónRESUMEN
Single myocytes were obtained from guinea-pig ventricles, and the catecholamine-induced Cl- current (ICl) was recorded by the patch-clamp method combined with the use of caged adenosine 3',5'-cyclic monophosphate (cAMP) and the concentration jump technique. The rapid bath application of isoprenaline was followed by a marked latency of approximately 1.7 s before a sigmoidal onset of the ICl activation, while the photolysis of caged cAMP was followed by no measurable latency. The ICl conductance was increased by increasing the concentration of caged cAMP with a threshold of approximately 5 microM and a one-half effective concentration of 25 microM. On the other hand, the rate of the activation increased in proportion to the logarithm of the concentration of caged cAMP. When the ultraviolet flash was repeated or the myocytes were pretreated with either forskolin, 3-isobutyl-1-methylxanthine, or okadaic acid, the rate of the ICl activation accelerated, and the whole time course of the ICl activation became monoexponential. These findings suggest that both the production of cAMP and reactions on the multiple phosphorylation sites of the channel protein are major rate-limiting steps in the ICl activation.