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1.
Nat Commun ; 15(1): 5353, 2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38918403

RESUMEN

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.


Asunto(s)
Neuronas , Nociceptina , Péptidos Opioides , Receptores Opioides , Animales , Péptidos Opioides/metabolismo , Receptores Opioides/metabolismo , Receptores Opioides/genética , Neuronas/metabolismo , Humanos , Ratones , Masculino , Área Tegmental Ventral/metabolismo , Receptor de Nociceptina , Células HEK293 , Encéfalo/metabolismo , Ratones Endogámicos C57BL , Ligandos , Técnicas Biosensibles/métodos
2.
Cell Calcium ; 117: 102834, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38006628

RESUMEN

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release-activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.


Asunto(s)
Benzamidas , Señalización del Calcio , Calcio , Pirazoles , Humanos , Señalización del Calcio/fisiología , Calcio/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Canales de Calcio/metabolismo , Proteína ORAI1/metabolismo
3.
Nat Methods ; 20(9): 1426-1436, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37474807

RESUMEN

Genetically encoded indicators engineered from G-protein-coupled receptors are important tools that enable high-resolution in vivo neuromodulator imaging. Here, we introduce a family of sensitive multicolor norepinephrine (NE) indicators, which includes nLightG (green) and nLightR (red). These tools report endogenous NE release in vitro, ex vivo and in vivo with improved sensitivity, ligand selectivity and kinetics, as well as a distinct pharmacological profile compared with previous state-of-the-art GRABNE indicators. Using in vivo multisite fiber photometry recordings of nLightG, we could simultaneously monitor optogenetically evoked NE release in the mouse locus coeruleus and hippocampus. Two-photon imaging of nLightG revealed locomotion and reward-related NE transients in the dorsal CA1 area of the hippocampus. Thus, the sensitive NE indicators introduced here represent an important addition to the current repertoire of indicators and provide the means for a thorough investigation of the NE system.


Asunto(s)
Locus Coeruleus , Norepinefrina , Animales , Ratones , Locus Coeruleus/fisiología , Hipocampo/fisiología , Receptores Acoplados a Proteínas G
4.
bioRxiv ; 2023 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-37292957

RESUMEN

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.

5.
Cell Calcium ; 105: 102616, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35792401

RESUMEN

In humans, there are three paralogs of the Orai Ca2+ channel that form the core of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still under active investigation, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating checkpoints among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the "ANSGA" nexus mutations in TM4 of human Orai1, which is reported to mimic the STIM1-activated state of the channel. In investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, that are not conserved among paralogs and that seem to be crucial for the channel activation triggered by the "ANSGA" mutations in Orai1. However, mutations of the same residues still allow gating of Orai1 by STIM1, suggesting that the ANSGA mutant of Orai1 may not be a surrogate for the STIM1-activated state of the Orai1 channel. Our results shed new light on these important gating checkpoints and show that the gating mechanism of Orai channels is affected by multiple factors that are not necessarily conserved among orai homologs, such as the TM4-TM3 coupling.


Asunto(s)
Canales de Calcio , Calcio , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Humanos , Mutación/genética , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo
7.
Nat Methods ; 19(2): 231-241, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35145320

RESUMEN

Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals.


Asunto(s)
Encéfalo/metabolismo , Imagen Molecular/métodos , Receptores de Orexina/genética , Orexinas/análisis , Proteínas Recombinantes/metabolismo , Animales , Conducta Animal , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Receptores de Orexina/metabolismo , Orexinas/genética , Orexinas/farmacología , Fotones , Proteínas Recombinantes/genética , Reproducibilidad de los Resultados , Sueño/fisiología
8.
Int J Mol Sci ; 21(16)2020 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-32764353

RESUMEN

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.


Asunto(s)
Compuestos de Boro/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genética , Moléculas de Interacción Estromal/genética , Animales , Compuestos de Boro/síntesis química , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteína ORAI1/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Molécula de Interacción Estromal 1/antagonistas & inhibidores , Moléculas de Interacción Estromal/antagonistas & inhibidores
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