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Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

Nature Cell Biology volume 5, pages 358–362 (2003)Cite this article

An Erratum to this article was published on 01 May 2003

Abstract

In chromaffin cells, exocytosis of single granules and properties of the fusion pore — the first connection between vesicular lumen and extracellular space1 — can be studied by cell-attached patch amperometry2,3, which couples patch-clamp capacitance measurements4,5,6,7 with simultaneous amperometric recordings of transmitter release8,9. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels10. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

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Figure 1: Capacitance steps evoked by applying calcium to an inside-out patch.
Figure 2: Statistical analysis of exocytotic activity.
Figure 3: Fusion pore analysis.
Figure 4: Endocytosis in excised inside-out patches.

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Acknowledgements

We thank L. Kwan for cell preparation and technical assistance. This work was supported by grants from the National Institutes of Health and the Nanobiotechnology Center (a Science and Technology Center programme of the NSF) to M.L., and a grant from the Ministerio de Educación y Cultura, Spain to G.A.d.T.

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Authors and Affiliations

  1. School of Applied and Engineering Physics, Cornell University, Ithaca, 14853-2501, New York, USA

    Gregor Dernick & Manfred Lindau

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

    Guillermo Alvarez de Toledo

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  1. Gregor Dernick
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Correspondence to Manfred Lindau.

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Dernick, G., Alvarez de Toledo, G. & Lindau, M. Exocytosis of single chromaffin granules in cell-free inside-out membrane patches. Nat Cell Biol 5, 358–362 (2003). https://doi.org/10.1038/ncb956

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