Protocol

Potassium Channels

Volume 491 of the series Methods in Molecular Biology pp 267-277

Rubidium Efflux as a Tool for the Pharmacological Characterisation of Compounds with BK Channel Opening Properties

  • Neil G. McKayAffiliated withBiomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam University
  • , Robert W. KirbyAffiliated withBiomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam University
  • , Kim LawsonAffiliated withBiomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam University

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Summary

This chapter describes a method of assaying rubidium (Rb+) efflux as a measure of potassium channel activity. In this assay, rubidium acts as a tracer for potassium movement across the cell membrane. HEK 293 cells expressing the alpha subunit of the human brain large-conductance, voltage-activated, calcium-sensitive potassium channel (BK channel) are loaded with Rb+, washed, and then incubated under experimental conditions. The cell supernatant is removed, and the remaining cell monolayer lysed. These two samples contain Rb+ that has moved out of the cell and Rb+ that remains in the cell, respectively. Measurement of the Rb+ content of these samples by flame atomic absorption spectrometry allows calculation of the percentage Rb+ efflux and, depending on the experimental design, provides pharmacological data about the control and test compounds used. In this chapter, we describe the protocol and steps for optimisation and illustrate this with data obtained using NS1619, a well-characterised BK channel opener.

Key words

Potassium channels BK channel Rubidium efflux Atomic absorption spectrometry Channel openers Small molecules Drug discovery