Skip to main content

Thallium Flux Assay for Measuring the Activity of Monovalent Cation Channels and Transporters

Part of the Methods in Molecular Biology book series (MIMB,volume 1684)

Abstract

Monovalent cation channels are critically important for physiological processes ranging from the control of neuronal excitability to the maintenance of solute balance. Mutations in these channels are associated with a multiplicity of diseases and monovalent cation channel-modulating drugs are used as therapeutics. Techniques that allow the measurement of the activity of these ion channels are useful for exploring their many biological roles as well as enabling the discovery and characterization of ion channel modulators for the purposes of drug discovery. Although there are numerous techniques for measuring the activity of monovalent cation channels, the thallium flux assay technique is a widely used fluorescence-based approach. Described herein is a method for using the thallium-flux technique for detecting and quantifying the activity of small-molecule potassium channel modulators in 384-well plates.

Key words

  • Ion channel
  • Potassium channel
  • Sodium channel
  • Drug discovery
  • High-throughput screening
  • Fluorescent dye
  • Thallium

This is a preview of subscription content, access via your institution.

Fig. 1

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Grynkiewicz G, Poenie M, Tsien RY (1984) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    Google Scholar 

  2. Minta A, Kao JPY, Tsien RY (1988) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264:8171–8178

    Google Scholar 

  3. Minta A, Tsien RY (1989) Fluorescent indicators for cytosolic sodium. J Biol Chem 264:19449–19457

    CAS  PubMed  Google Scholar 

  4. Hille B (1972) The permeability of the sodium channel to metal cations in myelinated nerve. J Gen Physiol 59:637–658

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Hille B (1973) Potassium channels in myelinated nerve. Selective permeability to small cations. J Gen Physiol 61:669–686

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Weaver CD, Harden D, Dworetzky SI, Robertson B, Knox RJ (2004) A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells. J Biomol Screen 9:671–677

    CAS  CrossRef  PubMed  Google Scholar 

  7. Niswender CM, Myers KA, Lou Q, Ayala J, Kim C, Conn PJ, Weaver CD (2008) Development of a novel and direct assay for high-throughput screening of Gi/o-linked GPCRs using thallium flux through GIRK channels. J Mol Pharm 73:1213–1224

    CAS  CrossRef  Google Scholar 

  8. Raphemot R, Lonergan DF, Nguyen TT, Utley T, Lewis LM, Kadakia R, Weaver CD, Gogliotti R, Hopkins C, Lindsley CW, Denton JS (2011) Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1. Front Pharmacol 2:75

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Yu H, Wu M, Townsend SD, Zou B, Long S, Daniels JS, McManus OB, Li M, Lindsley CW, Hopkins CR (2011) Discovery, synthesis, and structure activity relationship of a series of N-Aryl- bicyclo[2.2.1]heptane-2-carboxamides: characterization of ML213 as a novel KCNQ2 and KCNQ4 potassium channel opener. ACS Chem Nerosci 2:572–577

    CAS  CrossRef  Google Scholar 

  10. Lewis M, Bhave G, Chauder BA, Banerjee S, Lornsen K, Redha R, Fallen K, Lindsley CW, Weaver CD, Denton JS (2009) High-throughput screening reveals a small-molecule inhibitor of ROMK and Kir7.1. Mol Pharmacol 76:1094–1103

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Bhave G, Chauder BA, Liu W, Dawson ES, Kadakia R, Nguyen TT, Lewis LM, Meiler J, Weaver CD, Satlin LM, Lindsley CW, Denton JS (2010) Development of a selective small-molecule inhibitor of Kir1.1, the Renal Outer Medullary Potassium Channel. Mol Pharmacol 79:42–50

    CrossRef  PubMed  Google Scholar 

  12. Potet F, Lorinc AN, Chaigne S, Hopkins CR, Venkataraman R, Stepanovic SZ, Lewis LM, Days E, Sidorov VY, Engers DW, Zou B, Afshartous D, George AL Jr, Campbell CM, Balser JR, Li M, Baudenbacher FJ, Lindsley CW, Weaver CD, Kupershmidt S (2012) Identification and characterization of a compound that protects cardiac tissue from human ether-a-go-go-related gene (hERG)-related, drug-induced Arrhythmias. J Biol Chem 287:39613–39625

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  13. Li Q, Rottländer M, Xu M, Christoffersen CT, Frederiksen K, Wang MW, Jensen HS (2011) Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay. Anal Biochem 418:66–72

    CAS  CrossRef  PubMed  Google Scholar 

  14. Schmalhofer WA, Swensen AM, Thomas BS, Felix JP, Haedo RJ, Solly K, Kiss L, Kaczorowski GJ, Garcia ML (2010) A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-à-go-go related gene potassium channel. Assay Drug Dev Technol 8:714–726

    CAS  CrossRef  PubMed  Google Scholar 

  15. Delpire EJ, Days E, Lewis M, Mi D, Lindsley C, Weaver CD (2009) Small molecule screen identifies novel inhibitors of the neuronal K-Cl cotransporter KCC2. Proc Natl Acad Sci U S A 106:5383–5388

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  16. Carmosino M, Rizzo F, Torretta S, Procino G, Svelto M (2013) High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells. BMC Cell Biol 14:16

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  17. Di Virgilio F, Steinberg TH, Swanson JA, Silverstein SC (1988) J Immunol 140:915–920

    PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by the Vanderbilt Department of Pharmacology and the Vanderbilt Institute of Chemical Biology.

Conflict of Interest: CDW receives royalties from the sales of the Panoptic kinetic imaging plate reader, ThaLux Green, and the FLIPR Potassium Assay Kit. CDW is an owner of WaveFront Biosciences.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to C. David Weaver .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Weaver, C.D. (2018). Thallium Flux Assay for Measuring the Activity of Monovalent Cation Channels and Transporters. In: Shyng, SL., Valiyaveetil, F., Whorton, M. (eds) Potassium Channels. Methods in Molecular Biology, vol 1684. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7362-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7362-0_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7361-3

  • Online ISBN: 978-1-4939-7362-0

  • eBook Packages: Springer Protocols