High-Throughput Fluorescence Assays for Ion Channels and GPCRs

Vetter, Irina; Carter, David; Bassett, John J.; Deuis, Jennifer R.; Tay, Bryan; Jami, Sina; Robinson, Samuel D.

doi:10.1007/978-3-030-12457-1_3

Cited by 15 publications

(15 citation statements)

References 198 publications

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“…ACMA, acridine orange) 112,[126][127][128][129] or membrane potentials (e.g. oxonol VI, TMRE, VoltageFluors) [130][131][132][133][134] and do not directly report the translocated substrates.…”

Section: Rational Design Of Fluorescent Dyes To Follow Enzyme Functionmentioning

confidence: 99%

Current problems and future avenues in proteoliposome research

Amati

Graf

Deutschmann

et al. 2020

Biochemical Society Transactions

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Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

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Section: Rational Design Of Fluorescent Dyes To Follow Enzyme Functionmentioning

confidence: 99%

Current problems and future avenues in proteoliposome research

Amati

Graf

Deutschmann

et al. 2020

Biochemical Society Transactions

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“…Additionally, the Rgs20 gene (encoding a regulator of G q and G i protein signaling) is downregulated in OXYS rats relative to Wistar rats. The activation of the G q protein induces an increase in intracellular Ca 2+ concentration [56]; thus, the underexpression of its regulator may cause a decrease in intracellular Ca 2+ concentration. The observed change in gene expression may be regarded as a compensatory reaction designed to reduce the higher Ca 2+ concentration in the astrocytes.…”

Section: Genes Differentially Expressed Between Oxys and Wistar Rats ...mentioning

confidence: 99%

Changes in Glial Support of the Hippocampus during the Development of an Alzheimer’s Disease-like Pathology and Their Correction by Mitochondria-Targeted Antioxidant SkQ1

Rudnitskaya

Burnyasheva

Kozlova

et al. 2022

IJMS

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Astrocytes and microglia are the first cells to react to neurodegeneration, e.g., in Alzheimer’s disease (AD); however, the data on changes in glial support during the most common (sporadic) type of the disease are sparse. Using senescence-accelerated OXYS rats, which simulate key characteristics of sporadic AD, and Wistar rats (parental normal strain, control), we investigated hippocampal neurogenesis and glial changes during AD-like pathology. Using immunohistochemistry, we showed that the early stage of the pathology is accompanied by a lower intensity of neurogenesis and decreased astrocyte density in the dentate gyrus. The progressive stage is concurrent with reactive astrogliosis and microglia activation, as confirmed by increased cell densities and by the acquisition of cell-specific gene expression profiles, according to transcriptome sequencing data. Besides, here, we continued to analyze the anti-AD effects of prolonged supplementation with mitochondria-targeted antioxidant SkQ1. The antioxidant did not affect neurogenesis, partly normalized the gene expression profile of astrocytes and microglia, and shifted the resting/activated microglia ratio toward a decrease in the activated-cell density. In summary, both astrocytes and microglia are more vulnerable to AD-associated neurodegeneration in the CA3 area than in other hippocampal areas; SkQ1 had an anti-inflammatory effect and is a promising modality for AD prevention and treatment.

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“…For in vitro studies, investigations of long-term Ca 2+ dynamics are largely hindered by the cytotoxicity of GECIs or dyes (Rose et al, 2014;Smith et al, 2018). GCaMP-X is well suited to study Ca 2+ dynamics during neural development, or for high-throughput fluorescence assays to screen compounds with long-term effects (Vetter et al, 2020). For in vivo studies, due to the aforementioned concerns in GCaMP applications (Resendez et al, 2016), false-negative or falsepositive results by nuclear-filled GCaMP or under-expressed GCaMP are misleading especially in imaging large populations of neurons.…”

Section: Improved Neuron-compatibility Of Gcamp-xc and -Xnmentioning

confidence: 99%

Chronic Ca²⁺ imaging of cortical neurons with long-term expression of GCaMP-X

Geng

Tang

et al. 2022

Preprint

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Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. sensory response), and also mediate intracellular signaling cascades normally of longer time scales (e.g., Ca2+- dependent neuritogenesis). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate that the recently developed GCaMP-X outperforms GCaMP in long-term probe expression and/or chronic Ca2+ imaging. GCaMP-X shows much improved compatibility with neurons and thus more reliable than GCaMP as demonstrated in vivo by acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations over an extended time frame. Chronic Ca2+ imaging data (≥1 month) are acquired from the same set of cultured cortical neurons, unveiling that spontaneous/local Ca2+ activities would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length or soma size, the major metrics of oscillatory Ca2+, including rate, amplitude, synchrony among different neurons or organelles have also been examined along with the developmental stages. Both neuritogenesis and Ca2+ signals are dysregulated by GCaMP in virus-infected or transgenic neurons, in direct contrast to GCaMP-X without any noticeable side-effect. Such in vitro data altogether consolidate the unique importance of oscillatory Ca2+ to activity-dependent neuritogenesis, as one major factor responsible for the distinctions between GCaMP vs GCaMP-X in vivo. For the first time with GCaMP-X of long-term expression in neurons, spontaneous and sensory-evoked Ca2+ activities are imaged and evaluated both in vitro and in vivo, providing new opportunities to monitor neural development or other chronic processes concurrently with Ca2+ dynamics.

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High-Throughput Fluorescence Assays for Ion Channels and GPCRs

Cited by 15 publications

References 198 publications

Current problems and future avenues in proteoliposome research

Current problems and future avenues in proteoliposome research

Changes in Glial Support of the Hippocampus during the Development of an Alzheimer’s Disease-like Pathology and Their Correction by Mitochondria-Targeted Antioxidant SkQ1

Chronic Ca²⁺ imaging of cortical neurons with long-term expression of GCaMP-X

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High-Throughput Fluorescence Assays for Ion Channels and GPCRs

Cited by 15 publications

References 198 publications

Current problems and future avenues in proteoliposome research

Current problems and future avenues in proteoliposome research

Changes in Glial Support of the Hippocampus during the Development of an Alzheimer’s Disease-like Pathology and Their Correction by Mitochondria-Targeted Antioxidant SkQ1

Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

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Chronic Ca²⁺ imaging of cortical neurons with long-term expression of GCaMP-X