Skip to main content
SpringerLink
Log in

Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H+-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the KD-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters.

ᅟ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Decoursey TE. The voltage-gated proton channel: a riddle, wrapped in a mystery, inside an enigma. Biochemistry. 2015;54:3250–68.

    Article  CAS  PubMed  Google Scholar 

  2. Decoursey TE. Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the HV family. Physiol Rev. 2013;93:599–652.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Musset B, DeCoursey T. Biophysical properties of the voltage-gated proton channel Hv1. WIREs Membr Transp Signal. 2012;1:605–20.

    Article  CAS  Google Scholar 

  4. DeCoursey TE, Cherny VV. Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils. Biophys J. 1993;65:1590–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cherny VV, Murphy R, Sokolov V, Levis RA, DeCoursey TE. Properties of single voltage-gated proton channels in human eosinophils estimated by noise analysis and by direct measurement. J Gen Physiol. 2003;121:615–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lee S-Y, Letts JA, MacKinnon R. Functional reconstitution of purified human Hv1 H+ channels. J Mol Biol. 2009;387:1055–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Li Q, Shen R, Treger JS, Wanderling SS, Milewski W, Siwowska K, et al. Resting state of the human proton channel dimer in a lipid bilayer. Proc Natl Acad Sci U S A. 2015;112:E5926–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang J, Feng Y, Forgac M. Proton conduction and bafilomycin binding by the V0 domain of the coated vesicle V-ATPase. J Biol Chem. 1994;269:23518–23.

    CAS  PubMed  Google Scholar 

  9. Kemmer GC, Bogh SA, Urban M, Palmgren MG, Vosch T, Schiller J, et al. Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems. Analyst. 2015;140:6313–20.

    Article  CAS  PubMed  Google Scholar 

  10. Schwamborn M, Schumacher J, Sibold J, Teiwes NK, Steinem C. Monitoring ATPase induced pH changes in single proteoliposomes with the lipid-coupled fluorophore Oregon green 488. Analyst. 2017;142:2670–7.

    Article  CAS  PubMed  Google Scholar 

  11. Veshaguri S, Christensen SM, Kemmer GC, Ghale G, Møller MP, Lohr C, et al. Direct observation of proton pumping by a eukaryotic P-type ATPase. Science. 2016;351:1469–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hong L, Pathak MM, Kim IH, Ta D, Tombola F. Voltage-sensing domain of voltage-gated proton channel Hv1 shares mechanism of block with pore domains. Neuron. 2013;77:274–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Asuaje A, Smaldini P, Martin P, Enrique N, Orlowski A, Aiello EA, et al. The inhibition of voltage-gated H+ channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis. Pflugers Arch - Eur J Physiol. 2017;469:251–61.

    Article  CAS  Google Scholar 

  14. Schägger H, Vonjagow G. Tricine sodium dodecyl-sulfate polyacrylamide-gel electrophoresis for the separation of proteins in the range from 1-Kda to 100-Kda. Anal Biochem. 1987;166:368–79.

    Article  PubMed  Google Scholar 

  15. Han J, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev. 2010;110:2709–28.

    Article  CAS  PubMed  Google Scholar 

  16. Kozuch J, Steinem C, Hildebrandt P, Millo D. Combined electrochemistry and surface-enhanced infrared absorption spectroscopy of gramicidin A incorporated into tethered bilayer lipid membranes. Angew Chem Int Ed. 2012;51:8114–7.

    Article  CAS  Google Scholar 

  17. Kozuch J, Weichbrodt C, Millo D, Giller K, Becker S, Hildebrandt P, et al. Voltage-dependent structural changes of the membrane-bound anion channel hVDAC1 probed by SEIRA and electrochemical impedance spectroscopy. Phys Chem Chem Phys. 2014;16:9546–55.

    Article  CAS  PubMed  Google Scholar 

  18. Wiebalck S, Kozuch J, Tzschucke C, Jeuken LIC, Hildebrandt P. A novel tethered bilayer lipid membrane tailored for the IR spectroscopic investigation of membrane proteins: monitoring catalysis of cytochrome cbo3. J Phys Chem B. 2016;120:2249–56.

    Article  CAS  PubMed  Google Scholar 

  19. Rostovtseva TK, Aguilella VM, Vodyanoy I, Bezrukov SM, Parsegian VA. Membrane surface-charge titration probed by gramicidin A channel conductance. Biophys J. 1998;75:1783–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sandén T, Salomonsson L, Brzezinski P, Widengren J. Surface-coupled proton exchange of a membrane-bound proton acceptor. Proc Natl Acad Sci U S A. 2010;107:4129–34.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Cornelius F. Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics. Biochemistry. 2001;40:8842–51.

    Article  CAS  PubMed  Google Scholar 

  22. Rottenberg H, Moreno-Sanchez R. The proton pumping activity of H+-ATPases: an improved fluorescence assay. Biochim Biophys Acta Bioenerg. 1993;1183:161–70.

    Article  CAS  Google Scholar 

  23. Casadio R. Measurements of transmembrane pH differences of low extents in bacterial chromatophores. Eur Biophys J. 1991;19:189–201.

    Article  CAS  Google Scholar 

  24. Schuldiner S, Rottenberg H, Avron M. Determination of delta-pH in chloroplasts. 2. fluorescent amines as a probe for the determination of delta-pH in chloroplasts. Eur J Biochem. 1972;25:64-70.

    Article  CAS  PubMed  Google Scholar 

  25. D'Alessandro M, Turina P, Melandri BA. Quantitative evaluation of the intrinsic uncoupling modulated by ADP and Pi in the reconstituted ATP synthase of Escherichia coli. Biochim Biophys Acta. 2011;1807:130–43.

    Article  CAS  PubMed  Google Scholar 

  26. Branden M, Sanden T, Brzezinski P, Widengren J. Localized proton microcircuits at the biological membrane-water interface. Proc Natl Acad Sci U S A. 2006;103:19766–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. DeCoursey TE. Voltage-gated proton channels. Cell Mol Life Sci. 2008;65:2554–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Musset B, Cherny VV, Morgan D, Okamura Y, Ramsey IS, Clapham DE, et al. Detailed comparison of expressed and native voltage-gated proton channel currents. J Physiol. 2008;586:2477–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. DeCoursey TE. Voltage-gated proton channels: what's next? J Physiol. 2008;586:5305–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ramsey IS, Mokrab Y, Carvacho I, Sands ZA, Sansom MSP, Clapham DE. An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1. Nat Struct Mol Biol. 2010;17:869–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Seredenina T, Demaurex N, Krause K-H. Voltage-gated proton channels as novel drug targets: from NADPH oxidase regulation to sperm biology. Antioxid Redox Signal. 2015;23:490–513.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tombola F, Ulbrich MH, Isacoff EY. The voltage-gated proton channel Hv1 has two pores, each controlled by one voltage sensor. Neuron. 2008;58:546–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hong L, Kim IH, Tombola F. Molecular determinants of Hv1 proton channel inhibition by guanidine derivatives. Proc Natl Acad Sci U S A. 2014;111:9971–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Prof. Dr. T. Friedrich for the Hv1 cDNA. We thank J. Gerber-Nolte for technical support.

Funding

This study received financial support from the VW-foundation and the DFG (EXC314).

Author information

Authors and Affiliations

  1. Institut für Organische und Biomolekulare Chemie, Universität Göttingen, Tammannstr. 2, 37077, Göttingen, Germany

    Benjamin Gerdes, Rebecca M. Rixen, Kristina Kramer & Claudia Steinem

  2. Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 10623, Berlin, Germany

    Enrico Forbrig & Peter Hildebrandt

  3. Max-Planck-Institut für Dynamik und Selbstorganisation, Am Fassberg 11, 37077, Göttingen, Germany

    Claudia Steinem

Authors
  1. Benjamin Gerdes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca M. Rixen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristina Kramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Enrico Forbrig
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Hildebrandt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Claudia Steinem
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Claudia Steinem.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(PDF 833 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gerdes, B., Rixen, R.M., Kramer, K. et al. Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay. Anal Bioanal Chem 410, 6497–6505 (2018). https://doi.org/10.1007/s00216-018-1248-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-018-1248-7

Keywords

Search

Navigation