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Patch amperometry: high-resolution measurements of single-vesicle fusion and release

Nature Methods volume 2, pages 699–708 (2005)Cite this article

Patch amperometry is a new technique for the observation of single-vesicle exocytosis. Exocytosis of single vesicles as small as 50 nm in diameter can be detected by cell-attached patch-clamp admittance measurements1,2,3,4 indicating fusion of vesicles with the plasma membrane or by amperometry with a carbon fiber electrode (CFE)5,6,7,8,9 indicating release of oxidizable molecules such as catecholamines. The admittance measurement provides the membrane capacitance that increases in proportion to the membrane area because of the incorporation of the vesicle into the patch membrane. It also reveals the fusion pore conductance during an exocytotic event, giving an estimate of fusion pore dimensions. Amperometry provides the amount and time course of release of molecules that are readily oxidizable such as dopamine, norepinephrine or serotonin. This technique is capable of detecting as little as a few thousand molecules8,9. It also resolves the flux of catecholamines through a narrow fusion pore in a so-called foot signal that precedes rapid release indicated by an amperometric spike6. Patch amperometry combines high-resolution patch capacitance measurements with amperometry by placing the amperometric detector inside the patch pipet10. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines10,11,12,13,14. Thus, it is a unique tool to investigate the mechanisms that modulate quantal size and the effect of molecular manipulations affecting the properties of the fusion pore. Here we provide step-by-step instructions for the application of this method.

  • Cells to be studied plated onto 12 mm–diameter coverslips (for example, bovine chromaffin cells prepared as described15)

  • Bath and pipet solutions appropriate for cell type and experimental conditions to be studied. Bath solution (for chromaffin cells): 140 mM NaCl, 5 KCl mM, 5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-NaOH, 10 mM glucose (pH 7.3). Pipet solution (for chromaffin cells): 50 mM NaCl, 100 mM tetraethylammonium ion (TEA)-Cl, 5 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-NaOH (pH 7.3)

  • Sticky wax (Sticky Wax; Kerr Inc.)

  • 3 M KCl

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Figure 1: Patch amperometry setup.
Figure 2: Carbon fiber electrodes.
Figure 3: Patch amperometry pipet holders.
Figure 4: Patch amperometry recording from a rat chromaffin cell.

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Acknowledgements

We thank L. Kwan and J. Lenz for the cell preparations and excellent technical assistance; M. Montesinos, R. Borges, R. Staal and D. Sulzer for critical reading of the manuscript and for contributing information on using the Axopatch 200B for patch amperometry. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the National Institutes of Health (R01 NS38200) and the Nanobiotechnology Center (a Science and Technology Center (STC) program of the National Science Foundation, Agreement No. ECS-9876771) to M.L., and a grant from the Ministerio de Educación y Cultura, Spain to G.A.d.T.

Author information

Author notes

  1. Gregor Dernick

    Present address: F. Hoffmann-La Roche Ltd., CH-4070, Basel, Switzerland

  2. Liang-Wei Gong

    Present address: Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, 06510, USA

Authors and Affiliations

  1. School of Applied and Engineering Physics, Cornell University, Ithaca, 14850, New York, USA

    Gregor Dernick, Liang-Wei Gong & Manfred Lindau

  2. Department of Physiology and Biophysics, School of Medicine, University of Seville, Seville, E-41009, Spain

    Lucia Tabares & Guillermo Alvarez de Toledo

Authors
  1. Gregor Dernick
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  2. Liang-Wei Gong
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  3. Lucia Tabares
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  4. Guillermo Alvarez de Toledo
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  5. Manfred Lindau
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Corresponding author

Correspondence to Manfred Lindau.

Supplementary information

Supplementary Figure 1

Circuit diagram of remote controlled signal adder to switch sine wave stimulus on and off. (PDF 47 kb)

Supplementary Figure 2

Technical drawing of manual patch amperometry electrode holder. (PDF 138 kb)

Supplementary Note 1

Programmable CFE puller. (PDF 899 kb)

Supplementary Software (ZIP 97 kb)

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Dernick, G., Gong, LW., Tabares, L. et al. Patch amperometry: high-resolution measurements of single-vesicle fusion and release. Nat Methods 2, 699–708 (2005). https://doi.org/10.1038/nmeth0905-699

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