High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis

Abstract

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (Cm), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution Cm measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk Cm changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of Cm with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several Cm measurements can be performed daily.

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Figure 1: Hardware setup and minimal equivalent electrical circuits.
Figure 2: Whole-cell patch-clamp measurements of passive cell membrane parameters.
Figure 3: High-resolution cell-attached patch-clamp measurements of Cm reveal unitary exocytic events.

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Acknowledgements

This work was supported by the Slovenian Research Agency under grant no. P3 310 and project nos. J3 4051, J3-4146 and J3-3632. We acknowledge F. Henigman, M. Tester, G. Thiel, H.H. Chowdhury, J. Graf, S. Kinnamon, T. Pangršič, M. Rupnik and G. Zupančič for contributions to the development of the protocol as outlined in the published papers.

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B.R. and A.F. performed the experiments, analyzed the data and prepared the primary melanotroph and lactotroph cultures. B.R. and A.G. prepared the figures. J.J., M.K. and R.Z. developed the protocols for Cm measurements. All authors wrote the paper and discussed the results and implications and commented on the manuscript at all stages.

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Correspondence to Robert Zorec.

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Competing interests

B.R., A.G., J.J., A.F. and M.K. declare no competing financial interests. R.Z. has an equity interest in Celica Biomedical Center.

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Rituper, B., Guček, A., Jorgačevski, J. et al. High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis. Nat Protoc 8, 1169–1183 (2013). https://doi.org/10.1038/nprot.2013.069

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