Zusammenfassung
Protocol
Published in association with Cold Spring Harbor Laboratory Press
Nature Methods - 2, 699 - 708 (2005)
doi:10.1038/nmeth0905-699
Patch amperometry: high-resolution measurements of single-vesicle fusion and release
Gregor Dernick1, 3, Liang-Wei Gong1, 3, Lucia Tabares2, Guillermo Alvarez de Toledo2 & Manfred Lindau1
1 School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14850, USA.
2 Department of Physiology and Biophysics, School of Medicine, University of Seville, E-41009 Seville, Spain.
3 Present addresses: F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland (G.D.), and Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA (L.-W.G.).
Correspondence should be addressed to Manfred Lindau ml95@cornell.edu
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.
