A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential–Sensitive Dye and FLIPR

Vasilyev, Dmitry; Qin, Shan; Lee, Yan T.; Soloveva, Veronica; Nawoschik, Stanley; Kaftan, Edward; Dunlop, John; Mayer, Scott C.; Bowlby, Mark R.

doi:10.1177/1087057109345526

Cited by 19 publications

(9 citation statements)

References 32 publications

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“…Recently, such methods have been set up [140, 149] allowing to search for molecules, structurally unrelated to known modulators. By this way compound 11 has been discovered [139]; in addition, the HCN blocking properties of some known substances (CP-339,818 and loperamide) have been disclosed [150].…”

Section: Discussionmentioning

confidence: 99%

“…The blockade was use-independent; the binding site was suggested to be in the extracellular region of the channel. Loperamide and CP-339,818 [148], a K + -channel modulator evaluated in the same screening program [149], were tested on recombinant HCN1 and HCN4 expressed in HEK293 cells. Both compounds were 2-3 times more potent on HCN1 than on HCN4.…”

Section: Substances That Have Been Shown To Modulate Hcn Channel Actimentioning

confidence: 99%

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HCN Channels Modulators: The Need for Selectivity

Romanelli

Sartiani²,

Masi³

et al. 2016

CTMC

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Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, the molecular correlate of the hyperpolarization-activated current (I f /I h ), are membrane proteins which play an important role in several physiological processes and various pathological conditions. In the Sino Atrial Node (SAN) HCN4 is the target of ivabradine, a bradycardic agent that is, at the moment, the only drug which specifically blocks I f . Nevertheless, several other pharmacological agents have been shown to modulate HCN channels, a property that may contribute to their therapeutic activity and/or to their side effects.HCN channels are considered potential targets for developing drugs to treat several important pathologies, but a major issue in this field is the discovery of isoform-selective compounds, owing to the wide distribution of these proteins into the central and peripheral nervous systems, heart and other peripheral tissues. This survey is focused on the compounds that have been shown, or have been designed, to interact with HCN channels and on their binding sites, with the aim to summarize current knowledge and possibly to unveil useful information to design new potent and selective modulators.

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Section: Discussionmentioning

confidence: 99%

Section: Substances That Have Been Shown To Modulate Hcn Channel Actimentioning

confidence: 99%

HCN Channels Modulators: The Need for Selectivity

Romanelli

Sartiani²,

Masi³

et al. 2016

CTMC

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“…For this purpose, HL-1 cells were cultured in 96-well plates, loaded with the fluorescent membrane potential–sensitive indicator DiBAC 4 (3), and stimulated with carbachol. Membrane potential–sensitive dyes have been used in drug discovery for a wide variety of ion channels, including K + , 22,23 Na + , 24,25 hyperpolarization-activated cyclic-nucleotide gated, 26 and transient receptor potential 27 channels. Although fluorescent oxonol dyes, such as DiBAC 4 (3), provide a convenient and inexpensive approach for measuring changes in membrane potential, they are limited by their slow response compared with electrophysiological measurements.…”

Section: Discussionmentioning

confidence: 99%

A Real-Time Screening Assay for GIRK1/4 Channel Blockers

Walsh

2010

SLAS Discovery

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The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF.

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“…Additionally, there are currently no automated patch clamp platforms for assessing mechanical stimuli on DRG neurons. However, several ion channels important in pain pathologies have been studied in cell lines using this technique including Na V s, hyperpolarization activated cyclic nucleotide gated (HCN) and voltage-gated Ca 2+ channels (Ca V s) ( Payne et al, 2015 , Swensen et al, 2012 , Vasilyev et al, 2009 ). Overall, the relatively high throughput of these platforms makes them very useful for compound screening, but further development and cost optimization is necessary before automated patch clamp platforms replace the manual patch clamper in the lab.…”

Section: In Vitro Models To Study Peripheral Mechanisms Of Amentioning

confidence: 99%