Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and their Use in Ion Channel Screening Assays

Wolff, Christian; Fuks, B. B.; Chatelain, Pierre

doi:10.1177/1087057103257806

Cited by 109 publications

(80 citation statements)

References 27 publications

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“…30,31 Removal of media and/or washing the cells is also required during dye loading of commercial dyes used previously, which increases assay time and may promote cell detachment from the microplate wells. 31,32 This assay has a number of advantages as a rapid screen for assessing ligand potency and efficacy at µ-opioid receptors. It is rapid and real-time, it only requires the addition of a single reagent, and it is performed in intact cells.…”

Section: Discussionmentioning

confidence: 99%

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

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Section: Discussionmentioning

confidence: 99%

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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“…The lipophilic, anionic, bis-oxonol dye in the fluorometric imaging plate reader (FLIPR) membrane potential assay kit (Molecular Devices, Sunnyvale, CA) and fura-2 was used in dissociated VMN neurons to assess simultaneous fluorescent imaging of glucose-induced changes in membrane potential and intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) flux. The distribution across the plasma membrane of FLIPR dye is dependent on the membrane potential of the cell (32). Depolarization causes dye entry, where it binds to intracellular proteins and lipids, producing increased fluorescence signal.…”

Section: Methodsmentioning

confidence: 99%

“…Depolarization causes dye entry, where it binds to intracellular proteins and lipids, producing increased fluorescence signal. Hyperpolarization causes the dye to exit the cell, which decreases fluorescence (32). The VMN neurons were attached to coverslips, Excitations were set to 548 nm for membrane potential and 340 -380 nm for Ca 2ϩ .…”

Section: Methodsmentioning

confidence: 99%

Glucokinase Is a Critical Regulator of Ventromedial Hypothalamic Neuronal Glucosensing

et al. 2006

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To test the hypothesis that glucokinase is a critical regulator of neuronal glucosensing, glucokinase activity was increased, using a glucokinase activator drug, or decreased, using RNA interference combined with calcium imaging in freshly dissociated ventromedial hypothalamic nucleus (VMN) neurons or primary ventromedial hypothalamus (VMH; VMN plus arcuate nucleus) cultures. To assess the validity of our approach, we first showed that glucoseinduced (0.5-2.5 mmol/l) changes in intracellular Ca U nlike most neurons in the brain that utilize glucose to fuel their metabolic needs (1), a select group of neurons use glucose as a signaling molecule to alter their firing rate as a means of glucosensing (2,3). Glucose-excited neurons increase, whereas glucose-inhibited neurons decrease, their firing rate as ambient glucose levels rise (2-7). During situations of low glucose availability, glucose-inhibited neurons are activated and glucose-excited neurons inactivated (4 -8).The ventromedial hypothalamus (VMH) area contains both the ventromedial hypothalamic nucleus (VMN) and arcuate nucleus. Both contain glucosensing neurons that respond to differing levels of glucose and are linked to pathways involved in the regulation of glucose homeostasis (3-11) and the counterregulatory responses to hypoglycemia (12-19). Our work (6,7) and that of others (8) strongly support a role for glucokinase (hexokinase IV) as a key regulator of neuronal glucosensing, which is similar to its purported role in pancreatic ␤-cell glucosensing (20,21). We previously demonstrated that inhibition of glucokinase activity reduced glucose-excited and increased glucose-inhibited neuronal activity at 2.5 mmol/l glucose, the concentration at which they are normally active and inactive, respectively (6,7).Recurrent hypoglycemia is common in patients with type 1 diabetes, especially in children (22)(23)(24)(25). This leads to hypoglycemia-associated autonomic failure, in which counterregulatory responses to subsequent bouts of hypoglycemia are severely blunted (26 -28). Our previous studies suggested that the development of hypoglycemiaassociated autonomic failure might be associated with changes in the ability of VMH neurons to sense and respond to glucose (6,28). Furthermore, its development is associated with upregulation of glucokinase mRNA in the VMH (6,28,29). This upregulation might be a compensatory response that makes glucosensing neurons more sensitive to glucose by shifting their concentration-response to the left. If so, this could underlie the development of hypoglycemia-associated autonomic failure because it would require lower levels of glucose to be reached before counterregulatory responses were initiated. This would predict that increasing glucokinase activity would produce a leftward shift in glucose sensitivity in VMH glucosensing neurons such as it does in pancreatic ␤-cells, using a drug that increases glucokinase activity (30). It would also predict that reducing glucokinase activity would inhibit the response to glucose. Here, we ...

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“…Membrane potential sensitive dyes have been used to screen for ion channel inhibitors (Whiteaker et al, 2001;Wolff et al, 2003;Joesch et al, 2008) and have more recently also been used to screen for transporter inhibitors (Benjamin et al, 2005;Ruggiero et al, 2012). Here, we established the FLIPR system as a suitable assay to screen large compound libraries and to characterize amino acid transport inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Cheng

Shah

Bröer

et al. 2017

British J Pharmacology

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BACKGROUND AND PURPOSEThe neutral amino acid transporter B 0 AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B 0 AT1 mediates the Na + -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B 0 AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B 0 AT1. EXPERIMENTAL APPROACHA CHO-based cell line was generated, stably expressing collectrin and B 0 AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B 0 AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B 0 AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter. KEY RESULTSWe characterized a series of novel inhibitors of the B 0 AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC 50 of 44 ± 9 μM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine. CONCLUSION AND IMPLICATIONSThe tools established in this study can be widely used to identify new transport inhibitors. Using these tools, we were able to identify compounds that can be used to study epithelial transport, to induce protein restriction, or be developed further through medicinal chemistry. AbbreviationsCHO-BC, CHO cells stably transfected with B 0 AT1 and collectrin; FGF21, fibroblast growth factor 21; GIP, gastric inhibitory peptide; GLP-1, glucagon-like peptide 1; MW, molecular weight; NMDG, N-methyl-D-glucamine

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Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and their Use in Ion Channel Screening Assays

Cited by 109 publications

References 27 publications

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Glucokinase Is a Critical Regulator of Ventromedial Hypothalamic Neuronal Glucosensing

Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

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Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and their Use in Ion Channel Screening Assays

Cited by 109 publications

References 27 publications

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Glucokinase Is a Critical Regulator of Ventromedial Hypothalamic Neuronal Glucosensing

Identification of novel inhibitors of the amino acid transporter B0AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

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Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes