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Single-Molecule Analysis of Membrane Transporter Activity by Means of a Microsystem

  • Rikiya Watanabe
  • Naoki Soga
  • Shin-ya Ohdate
  • Hiroyuki Noji
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

Abstract

Emerging microtechnologies are aimed at developing a microsystem with densely packed array structure, i.e., an array with a femtoliter reaction chamber, for highly sensitive and quantitative biological assays. Here, we describe a novel femtoliter chamber array system (arrayed lipid bilayer chambers, ALBiC) that contains approximately a million femtoliter chambers, each sealed with a phospholipid bilayer membrane with extremely high efficiency (>90%). This novel platform enables detection of membrane transporter activity at the single-molecule level and thus expands the applicability of femtoliter chamber arrays to highly sensitive assays of transporters.

Key words

Microsystem Membrane transporter Single-molecule analysis 

Notes

Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research No. 15H05591 and 15H01312 to R.W. from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a Precursory Research for Embryonic Science (PRESTO) grant to R.W. from the Japan Science and Technology Agency.

References

  1. 1.
    Dunlop J, Bowlby M, Peri R, Vasilyev D, Arias R (2008) High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 7:358–368CrossRefPubMedGoogle Scholar
  2. 2.
    Zagnoni M (2012) Miniaturised technologies for the development of artificial lipid bilayer systems. Lab Chip 12:1026–1039CrossRefPubMedGoogle Scholar
  3. 3.
    Watanabe R, Soga N, Fujita D, Tabata KV, Yamauchi L, Hyeon Kim S, Asanuma D, Kamiya M, Urano Y, Suga H, Noji H (2014) Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity. Nat Commun 5:4519PubMedPubMedCentralGoogle Scholar
  4. 4.
    Watanabe R, Soga N, Yamanaka T, Noji H (2014) High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition. Sci Rep 4:7076CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Soga N, Watanabe R, Noji H (2015) Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters. Sci Rep 5:11025CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE (1996) Structure of staphylococcal a-hemolysin, a heptameric transmembrane pore. Science 274:1859–1866CrossRefPubMedGoogle Scholar
  7. 7.
    Junge W, Sielaff H, Engelbrecht S (2009) Torque generation and elastic power transmission in the rotary FOF1-ATPase. Nature 459:364–370CrossRefPubMedGoogle Scholar
  8. 8.
    Watanabe R, Tabata KV, Iino R, Ueno H, Iwamoto M, Oiki S, Noji H (2013) Biased Brownian stepping rotation of FoF1-ATP synthase driven by proton motive force. Nat Commun 4:1631CrossRefPubMedGoogle Scholar
  9. 9.
    Weber J (2010) Structural biology: toward the ATP synthase mechanism. Nat Chem Biol 6:794–795CrossRefPubMedGoogle Scholar
  10. 10.
    Yoshida M, Muneyuki E, Hisabori T (2001) ATP synthase—a marvellous rotary engine of the cell. Nat Rev Mol Cell Biol 2:669–677CrossRefPubMedGoogle Scholar
  11. 11.
    Asanuma D, Takaoka Y, Namiki S, Takikawa K, Kamiya M, Nagano T, Urano Y, Hirose K (2014) Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics. Angew Chem Int Ed Engl 53:6085–6089CrossRefPubMedGoogle Scholar
  12. 12.
    Iino R, Hasegawa R, Tabata KV, Noji H (2009) Mechanism of inhibition by C-terminal a-helices of the e subunit of Escherichia coli FoF1-ATP synthase. J Biol Chem 284:17457–17464CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Soga N, Kinosita K Jr, Yoshida M, Suzuki T (2011) Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase. FEBS J 278:2647–2654CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Soga N, Kinosita K Jr, Yoshida M, Suzuki T (2012) Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis. J Biol Chem 287:9633–9639CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Applied ChemistryGraduate School of Engineering, The University of TokyoTokyoJapan
  2. 2.PRESTO, JSTTokyoJapan

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