Dataset Information

A Quantitative Assay for Ca²⁺ Uptake through Normal and Pathological Hemichannels.

ABSTRACT: Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca²⁺) uptake with a Ca²⁺-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

SUBMITTER: Nardin C

PROVIDER: S-EPMC9266989 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Publications

A Quantitative Assay for Ca<sup>2+</sup> Uptake through Normal and Pathological Hemichannels.

Nardin Chiara C Tettey-Matey Abraham A Donati Viola V Marazziti Daniela D Di Pietro Chiara C Peres Chiara C Raspa Marcello M Zonta Francesco F Yang Guang G Gorelik Maryna M Singh Serena S Cardarelli Lia L Sidhu Sachdev S SS Mammano Fabio F

International journal of molecular sciences 20220630 13

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measureme ...[more]

PMID: 35806342

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Project description:Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Dataset Information

A Quantitative Assay for Ca²⁺ Uptake through Normal and Pathological Hemichannels.

Publications

A Quantitative Assay for Ca<sup>2+</sup> Uptake through Normal and Pathological Hemichannels.

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Dataset Information

A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.

Publications

A Quantitative Assay for Ca<sup>2+</sup> Uptake through Normal and Pathological Hemichannels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

A Quantitative Assay for Ca²⁺ Uptake through Normal and Pathological Hemichannels.