Высокопроизводительного скрининга для малых молекул модуляторы калиевых каналов входящего Rectifier
Summary
Методы разработку и утверждение количественного анализа флуоресценции для измерения активности калия внутрь выпрямителя (Кир) каналы высокой пропускной скрининга соединений представлены.
Abstract
Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay’s performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.
Protocol
Representative Results
Discussion
Обработка данных: После того как данные собраны, общий шаг анализа заключается в нормализации флуоресценции ответ каждого колодца, F, к своему исходному значению в начале эксперимента, F 0. Это обычно упоминается как "статическое отношение" и символизирует "F / F <s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Эта работа была поддержана финансирование от Национального института здоровья гранты 1R21NS073097-01 и 1R01DK082884 (JSD) и Фонд грантов PIER11VCTR Национальные институты.
Materials
| Name of the reagent | Company | Catalog number | Comments |
| pcDNA5/TO | Invitrogen | V1033-20 | Tetracycline-inducible expression vector |
| T-REx-HEK293 cells | Invitrogen | R71007 | Tetracycline-inducible cell line |
| Lipofectamine LTX/Plus Reagent | Invitrogen | 15338100 | Transfection reagent |
| FBS | ATLANTA Biologicals | S11550 | Cell culture media |
| DMEM | Invitrogen | 11965 | Cell culture media |
| Hygromycin B | Invitrogen | 10687-010 | Cell culture media |
| Blasticidin S | Invitrogen | R210-01 | Cell culture media |
| Penicillin/Streptomycin | Invitrogen | 15140 | Cell culture media |
| HBSS-divalent free | Mediatech | 21022CV | Cell washing |
| Trypsin-0.25% | Mediatech | 25053CI | Cell dissociation |
| Tetracycline-HCl | Sigma | T9823 | Induction reagent |
| Dialyzed FBS | ATLANTA Biologicals | S12650 | Plating media |
| FluoZin-2 | Invitrogen | F24189 | Fluorescent dye |
| Pluronic F-127 | Invitrogen | P-3000MP | Dye loading |
| HBSS | Invitrogen | 14175 | Assay buffer |
| HEPES | Invitrogen | 15630 | Assay buffer |
| NaHCO3 | Sigma | S6297 | Tl+ stimulus buffer |
| MgSO4 | Sigma | M2643 | Tl+ stimulus buffer |
| CaSO4•2H2O | Sigma | C3771 | Tl+ stimulus buffer |
| D-Glucose | Sigma | G7528 | Tl+ stimulus buffer |
| Thallium sulfate | Aldrich | 204625 | Tl+ stimulus buffer |
| HEPES | Sigma | H4034 | Tl+ stimulus buffer |
| DMSO | Sigma | D4540 | Solvent |
| Eight-channel electronic pipettor | Biohit | E300 | Cell plating in 384-well plates |
| BD PureCoat amine-coated 384-well plates | BD Biosciences | 356719 | Assay microplates |
| Echo qualified 384-Well polypropylene microplate (384PP) | Labcyte | P-05525 | Compound source microplates |
| 384-well polypropylene microplates | Greiner Bio-One | 781280 | |
| Multidrop Combi reagent dispenser | Thermo Scientific | 5840300 | |
| ELx405 microplate washer | BioTek | ELx405HT | Automated cell washing |
| Echo liquid handler | Labcyte | Labcyte Echo 550 | |
| Bravo automated liquid handling platform | Agilent Technologies | Standard model | |
| Hamamatsu FDSS 6000 | Hamamatsu | Kinetic imaging plate reader |
Table 1. List of Materials and Reagents.
References
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