Skip to main content
SpringerLink
Log in
Book cover

Calcium Signalling pp 65–73Cite as

Electrophysiological Characterization of Calcium-Permeable Channels Using Planar Lipid Bilayer

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1925))

Abstract

Numerous researchers tried to identify the key players of calcium signaling in mitochondria using molecular and cell biology techniques for more than five decades. However, only an integrated approach involving also electrophysiological techniques has finally allowed to define the components of the protein complex responsible for the uptake of this ion into mitochondria.

Here we describe the protocol used for the electrophysiological characterization of the mitochondrial calcium uniporter (MCU) complex: the following outline indicates step-by-step the setup of planar lipid bilayer experiments.

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

eBook
USD   44.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   59.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Vasington FD, Murphy JV (1962) Ca ion uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J Biol Chem 237:2670–2677

    CAS  PubMed  Google Scholar 

  2. Deluca HF, Engstrom GW (1961) Calcium uptake by rat kidney mitochondria. Proc Natl Acad Sci U S A 47:1744–1750

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Graier WF, Frieden M, Malli R (2007) Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375–396

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Starkov AA (2010) The molecular identity of the mitochondrial Ca2+ sequestration system. FEBS J 277:3652–3663

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. De Stefani D, Raffaello A, Teardo E et al (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476:336–340

    Article  PubMed Central  PubMed  Google Scholar 

  6. Baughman JM, Perocchi F, Girgis HS et al (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476:341–345

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Bick AG, Calvo SE, Mootha VK (2012) Evolutionary diversity of the mitochondrial calcium uniporter. Science 336:886–886

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Cheng Y, Perocchi F (2015) ProtPhylo: identification of protein–phenotype and protein–protein functional associations via phylogenetic profiling. Nucleic Acids Res 43:W160–W168

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Kirichok Y, Krapivinsky G, Clapham DE (2004) The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427:360–364

    Article  CAS  PubMed  Google Scholar 

  10. Vais H, Mallilankaraman K, Mak DD, Hoff H, Payne R, Tanis JE, Foskett JK (2016) EMRE is a matrix Ca(2+) sensor that governs gatekeeping of the mitochondrial Ca(2+) uniporter. Cell Rep 14(3):403–410

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Raffaello A, De Stefani D, Sabbadin D et al (2013) The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. EMBO J 32:2362–2376

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Patron M, Checchetto V, Raffaello A et al (2014) MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity. Mol Cell 53:726–737

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Csordás G, Golenár T, Seifert EL et al (2013) MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca2+ uniporter. Cell Metab 17:976–987

    Article  PubMed Central  PubMed  Google Scholar 

  14. Lam SS, Martell JD, Kamer KJ et al (2014) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12:51–54

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Hoffman NE, Chandramoorthy HC, Shamugapriya S et al (2013) MICU1 motifs define mitochondrial calcium uniporter binding and activity. Cell Rep 5:1576–1588

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Vecellio Reane D, Vallese F, Checchetto V et al (2016) A MICU1 splice variant confers high sensitivity to the mitochondrial Ca2+ uptake machinery of skeletal muscle. Mol Cell 64:760–773

    Article  CAS  PubMed  Google Scholar 

  17. Plovanich M, Bogorad RL, Sancak Y et al (2013) MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling. PLoS One 8:e55785

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Penna E, Espino J, De Stefani D, Rizzuto R (2018) The MCU complex in cell death. Cell Calcium 69:73–80

    Article  CAS  PubMed  Google Scholar 

  19. Mammucari C, Gherardi G, Rizzuto R (2017) Structure, activity regulation, and role of the mitochondrial calcium uniporter in health and disease. Front Oncol 10(7):139

    Article  Google Scholar 

  20. Carraretto L, Formentin E, Teardo E, Checchetto V, Tomizioli M, Morosinotto T, Giacometti GM, Finazzi G, Szabó I (2013) A thylakoid-located two-pore K+ channel controls photosynthetic light utilization in plants. Science 342(6154):114–118

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, University of Padua, Padua, Italy

    Vanessa Checchetto & Ildikò Szabò

Authors
  1. Vanessa Checchetto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ildikò Szabò
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vanessa Checchetto .

Editor information

Editors and Affiliations

  1. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Anna Raffaello

  2. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Denis Vecellio Reane

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Checchetto, V., Szabò, I. (2019). Electrophysiological Characterization of Calcium-Permeable Channels Using Planar Lipid Bilayer. In: Raffaello, A., Vecellio Reane, D. (eds) Calcium Signalling. Methods in Molecular Biology, vol 1925. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9018-4_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9018-4_6

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9017-7

  • Online ISBN: 978-1-4939-9018-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation