Advertisement

Developing High-Throughput Assays to Analyze and Screen Electrophysiological Phenotypes

  • Jen Q. Pan
  • David Baez-Nieto
  • Andrew Allen
  • Hao-Ran Wang
  • Jeffrey R. Cottrell
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1787)

Abstract

Ion channels represent nearly a quarter of all targets that currently available medications modulate, and their dysfunction underlies increasing number of human diseases. Functional analysis of ion channels have traditionally been a bottleneck in large-scale analyses. Recent technological breakthroughs in automated planar electrophysiology have democratized the technique to enable high-throughput patch clamping at scale. In this chapter, we describe the methodology to perform a phenotypic screen on voltage-gated calcium channels across many different genetic coding variations and against small-molecule modulators. We first describe the procedures to establish inducible heterologous ion channel expression in HEK293 cells, where each cell incorporates one copy of a target protein cDNA—a step that is critical for producing stable and consistent expression of ion channels. We then describe the experimental and analytical methods for analyzing the function of ion channels using high-throughput planar electrophysiology.

Key words

Patch clamp Planar electrophysiology Ion channels Voltage-gated calcium channels High throughput Electrophysiology Phenotypic screen 

References

  1. 1.
    Gadsby DC, Vergani P, Csanady L (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440:477–483CrossRefPubMedGoogle Scholar
  2. 2.
    Szeliga MA, Hedley PL, Green CP, Moller DV, Christiansen M (2010) Long QT syndrome: a genetic cardiac channelopathy. Kardiol Pol 68:575–583Google Scholar
  3. 3.
    Litan A, Langhans SA (2015) Cancer as a channelopathy: ion channels and pumps in tumor development and progression. Front Cell Neurosci 9:86CrossRefPubMedGoogle Scholar
  4. 4.
    Cai B, Chen X, Liu F, Li J, Gu L, Liu JR et al (2014) A cell-based functional assay using a green fluorescent protein-based calcium indicator dCys-GCaMP. Assay Drug Dev Technol 12:342–351CrossRefPubMedGoogle Scholar
  5. 5.
    Kim JG, Park SW, Byun D, Choi WS, Sung DJ, Shin KC et al (2016) Fluid flow facilitates inward rectifier K+ current by convectively restoring [K+] at the cell membrane surface. Sci Rep 6:39585CrossRefPubMedGoogle Scholar
  6. 6.
    Beacham DW, Blackmer T, OG M, Hanson GT (2010) Cell-based potassium ion channel screening using the FluxOR assay. J Biomol Screen 15:441–446CrossRefGoogle Scholar
  7. 7.
    Milligan CJ, Moller C (2013) Automated planar patch-clamp. Methods Mol Biol 998:171–187CrossRefGoogle Scholar
  8. 8.
    Obergrussberger A, Bruggemann A, Goetze TA, Rapedius M, Haarmann C, Rinke I et al (2016) Automated patch clamp meets high-throughput screening: 384 Cells recorded in parallel on a planar patch clamp module. J Lab Autom 21:779–793CrossRefGoogle Scholar
  9. 9.
    Polonchuk L (2014) Industrializing electrophysiology: HT automated patch clamp on SyncroPatch(R) 96 using instant frozen cells. Methods Mol Biol 1183:81–92CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jen Q. Pan
    • 1
  • David Baez-Nieto
    • 1
  • Andrew Allen
    • 1
  • Hao-Ran Wang
    • 1
  • Jeffrey R. Cottrell
    • 1
  1. 1.Stanley Center for Psychiatric ResearchBroad Institute of MIT and HarvardCambridgeUSA

Personalised recommendations