Analytical and Bioanalytical Chemistry

, Volume 408, Issue 29, pp 8529–8538 | Cite as

Quantitative characterization of capsaicin-induced TRPV1 ion channel activation in HEK293 cells by impedance spectroscopy

  • Maxi Weyer
  • Heinz-Georg Jahnke
  • Dana Krinke
  • Franziska D. Zitzmann
  • Kerstin Hill
  • Michael Schaefer
  • Andrea A. Robitzki
Research Paper

Abstract

The analysis of receptor activity, especially in its native cellular environment, has always been of great interest to evaluate its intrinsic but also downstream biological activity. An important group of cellular receptors are ion channels. Since they are involved in a broad range of crucial cell functions, they represent important therapeutic targets. Thus, novel analytical techniques for the quantitative monitoring and screening of biological receptor activity are of great interest. In this context, we developed an impedance spectroscopy-based label-free and non-invasive monitoring system that enabled us to analyze the activation of the transient receptor potential channel Vanilloid 1 (TRPV1) in detail. TRPV1 channel activation by capsaicin resulted in a reproducible impedance decrease. Moreover, concentration response curves with an EC50 value of 0.9 μM could be determined. Control experiments with non TRPV1 channel expressing HEK cells as well as experiments with the TRPV1 channel blocker ruthenium red validated the specificity of the observed impedance decrease. More strikingly, through correlative studies with a cytoskeleton restructuring inhibitor mixture and equivalent circuit analysis of the acquired impedance spectra, we could quantitatively discriminate between the direct TRPV1 channel activation and downstream-induced biological effects. In summary, we developed a quantitative impedimetric monitoring system for the analysis of TRPV1 channel activity as well as downstream-induced biological activity in living cells. It has the capabilities to identify novel ion channel activators as well as inhibitors for the TRPV1 channel but could also easily be applied to other ion channel-based receptors.

Keywords

TRPV1 channel activation monitoring Time-resolved quantification of ion channel activity Impedance spectroscopy Interdigital electrode arrays Equivalent circuit modeling 

Notes

Acknowledgments

This work was funded by the German Research Foundation (DFG; Graduate school Interneuro GRK 1097, and FOR 2177 InCheM RO 2652/1-1). Impedance analyzer, confocal microscope, and clean room equipment were funded by the Free State of Saxony and the European Union (SMWK/EFRE).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2016_9978_MOESM1_ESM.pdf (156 kb)
ESM 1 (PDF 156 kb)
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(AVI 2696 kb)

References

  1. 1.
    Pedersen SF, Owsianik G, Nilius B. TRP channels: an overview. Cell Calcium. 2005;38(3–4):233–52. doi: 10.1016/j.ceca.2005.06.028.CrossRefGoogle Scholar
  2. 2.
    Julius D. TRP channels and pain. Annu Rev Cell Dev Biol. 2013;29:355–84. doi: 10.1146/annurev-cellbio-101011-155833.CrossRefGoogle Scholar
  3. 3.
    Ishimaru Y, Matsunami H. Transient receptor potential (TRP) channels and taste sensation. J Dent Res. 2009;88(3):212–8. doi: 10.1177/0022034508330212.CrossRefGoogle Scholar
  4. 4.
    Dhaka A, Viswanath V, Patapoutian A. Trp ion channels and temperature sensation. Annu Rev Neurosci. 2006;29:135–61. doi: 10.1146/annurev.neuro.29.051605.112958.CrossRefGoogle Scholar
  5. 5.
    Rosenbaum T, Simon SA. TRPV1 Receptors and Signal Transduction. In: Liedtke WB, Heller S, editors. TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. Frontiers in Neuroscience. Boca Raton (FL) 2007.Google Scholar
  6. 6.
    Cortright DN, Szallasi A. Biochemical pharmacology of the vanilloid receptor TRPV1. An update. Eur J Biochem. 2004;271(10):1814–9. doi: 10.1111/j.1432-1033.2004.04082.x.CrossRefGoogle Scholar
  7. 7.
    Dussor G, Yan J, Xie JY, Ossipov MH, Dodick DW, Porreca F. Targeting TRP channels for novel migraine therapeutics. ACS Chem Neurosci. 2014;5(11):1085–96. doi: 10.1021/cn500083e.CrossRefGoogle Scholar
  8. 8.
    Moran MM, McAlexander MA, Biro T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov. 2011;10(8):601–20. doi: 10.1038/nrd3456.CrossRefGoogle Scholar
  9. 9.
    Lev S, Minke B. Constitutive activity of TRP channels methods for measuring the activity and its outcome. Methods Enzymol. 2010;484:591–612. doi: 10.1016/B978-0-12-381298-8.00029-0.CrossRefGoogle Scholar
  10. 10.
    Sunesen M, Jacobsen RB. Study of TRP channels by automated patch clamp systems. In: Islam SM, editor. Transient receptor potential channels. Dordrecht: Springer; 2011. p. 107–23.CrossRefGoogle Scholar
  11. 11.
    Jahnke HG, Rothermel A, Sternberger I, Mack TG, Kurz RG, Panke O, et al. An impedimetric microelectrode-based array sensor for label-free detection of tau hyperphosphorylation in human cells. Lab Chip. 2009;9(10):1422–8. doi: 10.1039/b819754g.CrossRefGoogle Scholar
  12. 12.
    Haas S, Jahnke HG, Glass M, Azendorf R, Schmidt S, Robitzki AA. Real-time monitoring of relaxation and contractility of smooth muscle cells on a novel biohybrid chip. Lab Chip. 2010;10(21):2965–71. doi: 10.1039/c0lc00008f.CrossRefGoogle Scholar
  13. 13.
    Rahman AR, Register J, Vuppala G, Bhansali S. Cell culture monitoring by impedance mapping using a multielectrode scanning impedance spectroscopy system (CellMap). Physiol Meas. 2008;29(6):S227–39. doi: 10.1088/0967-3334/29/6/S20.CrossRefGoogle Scholar
  14. 14.
    te Kamp V, Lindner R, Jahnke HG, Krinke D, Kostelnik KB, Beck-Sickinger AG, et al. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells. Biosens Bioelectron. 2015;67:386–93. doi: 10.1016/j.bios.2014.08.066.CrossRefGoogle Scholar
  15. 15.
    Panke O, Weigel W, Schmidt S, Steude A, Robitzki AA. A cell-based impedance assay for monitoring transient receptor potential (TRP) ion channel activity. Biosens Bioelectron. 2011;26(5):2376–82. doi: 10.1016/j.bios.2010.10.015.CrossRefGoogle Scholar
  16. 16.
    Krinke D, Jahnke HG, Mack TG, Hirche A, Striggow F, Robitzki AA. A novel organotypic tauopathy model on a new microcavity chip for bioelectronic label-free and real time monitoring. Biosens Bioelectron. 2010;26(1):162–8. doi: 10.1016/j.bios.2010.06.002.CrossRefGoogle Scholar
  17. 17.
    Jahnke HG, Braesigk A, Mack TG, Ponick S, Striggow F, Robitzki AA. Impedance spectroscopy based measurement system for quantitative and label-free real-time monitoring of tauopathy in hippocampal slice cultures. Biosens Bioelectron. 2012;32(1):250–8. doi: 10.1016/j.bios.2011.12.026.CrossRefGoogle Scholar
  18. 18.
    Trapani JG, Korn SJ. Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines. BMC Neurosci. 2003;4:15. doi: 10.1186/1471-2202-4-15.CrossRefGoogle Scholar
  19. 19.
    Mergler S, Skrzypski M, Sassek M, Pietrzak P, Pucci C, Wiedenmann B, et al. Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells. Cell Signal. 2012;24(1):233–46. doi: 10.1016/j.cellsig.2011.09.005.CrossRefGoogle Scholar
  20. 20.
    Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997;389(6653):816–24. doi: 10.1038/39807.CrossRefGoogle Scholar
  21. 21.
    Basith S, Cui M, Hong S, Choi S. Harnessing the Therapeutic Potential of Capsaicin and Its Analogues in Pain and Other Diseases. Molecules. 2016; 21(8). doi: 10.3390/molecules21080966.
  22. 22.
    Geng S, Zheng Y, Meng M, Guo Z, Cao N, Ma X, et al. Gingerol reverses the cancer-promoting effect of capsaicin by increased TRPV1 level in a urethane-induced lung carcinogenic model. J Agric Food Chem. 2016. doi: 10.1021/acs.jafc.6b02480.Google Scholar
  23. 23.
    Fattori V, Hohmann MS, Rossaneis AC, Pinho-Ribeiro FA, Verri WA. Capsaicin: current understanding of its mechanisms and therapy of pain and other pre-clinical and clinical uses. Molecules. 2016; 21(7). doi: 10.3390/molecules21070844.
  24. 24.
    Dhaka A, Uzzell V, Dubin AE, Mathur J, Petrus M, Bandell M, et al. TRPV1 is activated by both acidic and basic pH. J Neurosci. 2009;29(1):153–8. doi: 10.1523/JNEUROSCI.4901-08.2009.CrossRefGoogle Scholar
  25. 25.
    Oyagbemi AA, Saba AB, Azeez OI. Capsaicin: a novel chemopreventive molecule and its underlying molecular mechanisms of action. Indian J Cancer. 2010;47(1):53–8. doi: 10.4103/0019-509X.58860.CrossRefGoogle Scholar
  26. 26.
    Kim S, Kang C, Shin CY, Hwang SW, Yang YD, Shim WS, et al. TRPV1 recapitulates native capsaicin receptor in sensory neurons in association with Fas-associated factor 1. J Neurosci. 2006;26(9):2403–12. doi: 10.1523/JNEUROSCI.4691-05.2006.CrossRefGoogle Scholar
  27. 27.
    Ponti J, Ceriotti L, Munaro B, Farina M, Munari A, Whelan M, et al. Comparison of impedance-based sensors for cell adhesion monitoring and in vitro methods for detecting cytotoxicity induced by chemicals. Altern Lab Anim. 2006;34(5):515–25.Google Scholar
  28. 28.
    Sarró E, Lecina M, Fontova A, Gòdia F, Bragós R, Cairó JJ. Real-time and on-line monitoring of morphological cell parameters using electrical impedance spectroscopy measurements. J Chem Technol Biotechnol. 2015:n/a-n/a. doi: 10.1002/jctb.4765.
  29. 29.
    Peng GE, Wilson SR, Weiner OD. A pharmacological cocktail for arresting actin dynamics in living cells. Mol Biol Cell. 2011;22(21):3986–94. doi: 10.1091/mbc.E11-04-0379.CrossRefGoogle Scholar
  30. 30.
    Eichler M, Jahnke HG, Krinke D, Muller A, Schmidt S, Azendorf R, et al. A novel 96-well multielectrode array based impedimetric monitoring platform for comparative drug efficacy analysis on 2D and 3D brain tumor cultures. Biosens Bioelectron. 2015;67:582–9. doi: 10.1016/j.bios.2014.09.049.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Maxi Weyer
    • 1
  • Heinz-Georg Jahnke
    • 1
  • Dana Krinke
    • 1
  • Franziska D. Zitzmann
    • 1
  • Kerstin Hill
    • 2
  • Michael Schaefer
    • 2
  • Andrea A. Robitzki
    • 1
  1. 1.Centre for Biotechnology and Biomedicine, Molecular Biological-Biochemical Processing TechnologyLeipzig UniversityLeipzigGermany
  2. 2.Rudolf-Boehm-Institute for Pharmacology and ToxicologyLeipzig UniversityLeipzigGermany

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