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Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies

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Potassium Channels

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

Abstract

Quantitative investigations into functional properties of purified ion channel proteins using standard electrophysiological methods are challenging, in particular for the determination of average ion channel behavior following rapid changes in experimental conditions (e.g., ligand concentration). Here, we describe a method for determining the functional activity of liposome-reconstituted K+ channels using a stopped-flow fluorometric ion flux assay. Channel activity is quantified by measuring the rate of fluorescence decrease of a liposome-encapsulated fluorophore, specifically quenched by thallium ions entering the liposomes via open channels. This method is well suited for studying the lipid bilayer dependence of channel activity, the activation and desensitization kinetics of ligand-dependent K+ channels, and channel modulation by channel agonists, blockers, or other antagonists.

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Acknowledgment

This work was supported by NIH grants R01 GM088352 (to C.M.N.) and R01 GM021342 (to O.S.A.), NIH fellowship F32GM087865 (to D.J.P.), and a Kellen Fellowship (R.R.).

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Authors and Affiliations

  1. Department of Anesthesiology, Weill Cornell Medical College, 1300 York Ave., New York, NY, 10065, USA

    David J. Posson & Crina M. Nimigean

  2. Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Ave., New York, NY, 10065, USA

    David J. Posson, Radda Rusinova, Olaf S. Andersen & Crina M. Nimigean

  3. Vertex Pharmaceuticals Inc., 50 Northern Ave., Boston, MA, 02210, USA

    David J. Posson

  4. Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave., New York, NY, 10065, USA

    Crina M. Nimigean

Authors
  1. David J. Posson
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  2. Radda Rusinova
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  3. Olaf S. Andersen
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  4. Crina M. Nimigean
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Corresponding author

Correspondence to Crina M. Nimigean .

Editor information

Editors and Affiliations

  1. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA

    Show-Ling Shyng

  2. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA

    Francis I. Valiyaveetil

  3. The Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA

    Matt Whorton

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Posson, D.J., Rusinova, R., Andersen, O.S., Nimigean, C.M. (2018). Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies. 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_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7362-0_17

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  • 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

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