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

Nature Protocols volume 8, pages 1169–1183 (2013)Cite this article

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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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References

  1. Grant, B.D. & Donaldson, J.G. Pathways and mechanisms of endocytic recycling. Nat. Rev. Mol. Cell Biol. 10, 597–608 (2009).

    Article  CAS  Google Scholar 

  2. Schweizer, F.E. & Ryan, T.A. The synaptic vesicle: cycle of exocytosis and endocytosis. Curr. Opin. Neurobiol. 16, 298–304 (2006).

    Article  CAS  Google Scholar 

  3. Rituper, B., Davletov, B. & Zorec, R. Lipid-protein interactions in exocytotic release of hormones and neurotransmitters. Clin. Lipidol. 5, 747–761 (2010).

    Article  CAS  Google Scholar 

  4. Katz, B. The Release of Neural Transmitter Substances (Liverpool University Press, 1969).

  5. Neher, E. & Sakmann, B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799–802 (1976).

    Article  CAS  Google Scholar 

  6. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100 (1981).

    Article  CAS  Google Scholar 

  7. Neher, E. & Marty, A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc. Natl. Acad. Sci. USA 79, 6712–6716 (1982).

    Article  CAS  Google Scholar 

  8. Zorec, R., Henigman, F., Mason, W. & Kordaš, M. Electrophysiological study of hormone secretion by single adenohypophyseal cells. Methods Neurosci. 4, 194–210 (1991).

    Article  Google Scholar 

  9. Lledo, P., Vernier, P., Vincent, J., Mason, W. & Zorec, R. Inhibition of Rab3B expression attenuates Ca2+-dependent exocytosis in rat anterior pituitary cells. Nature 364, 540–544 (1993).

    Article  CAS  Google Scholar 

  10. Stenovec, M., Kreft, M., Poberaj, I., Betz, W. & Zorec, R. Slow spontaneous secretion from single large dense-core vesicles monitored in neuroendocrine cells. FASEB J. 18, 1270–1272 (2004).

    Article  CAS  Google Scholar 

  11. Lindau, M. High-resolution electrophysiological techniques for the study of calcium-activated exocytosis. Biochim. Biophys. Acta 1820, 1234–1242 (2011).

    Article  Google Scholar 

  12. Zorec, R., Sikdar, S. & Mason, W. Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs. Direct evidence from changes in membrane capacitance. J. Gen. Physiol. 97, 473–497 (1991).

    Article  CAS  Google Scholar 

  13. Sikdar, S.K., Zorec, R., Brown, D. & Mason, W.T. Dual effects of G-protein activation on Ca-dependent exocytosis in bovine lactotrophs. FEBS Lett. 253, 88–92 (1989).

    Article  CAS  Google Scholar 

  14. Sikdar, S.K., Zorec, R. & Mason, W.T. cAMP directly facilitates Ca-induced exocytosis in bovine lactotrophs. FEBS Lett. 273, 150–154 (1990).

    Article  CAS  Google Scholar 

  15. Rupnik, M. & Zorec, R. Cytosolic chloride ions stimulate Ca2+-induced exocytosis in melanotrophs. FEBS Lett. 303, 221–223 (1992).

    Article  CAS  Google Scholar 

  16. Rupnik, M. et al. Increased cytosolic chloride affects depolarization-induced changes in membrane capacitance and cytosolic calcium activity in rat melanotrophs. Ann. N Y Acad. Sci. 710, 319–327 (1994).

    Article  CAS  Google Scholar 

  17. Ellis-Davies, G.C. & Kaplan, J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc. Natl. Acad. Sci. USA 91, 187–191 (1994).

    Article  CAS  Google Scholar 

  18. Thomas, P., Wong, J.G. & Almers, W. Millisecond studies of secretion in single rat pituitary cells stimulated by flash photolysis of caged Ca2+. EMBO J. 12, 303–306 (1993).

    Article  CAS  Google Scholar 

  19. Thomas, P., Wong, J.G., Lee, A.K. & Almers, W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 11, 93–104 (1993).

    Article  CAS  Google Scholar 

  20. Kreft, M. et al. The heterotrimeric Gi3 protein acts in slow but not in fast exocytosis of rat melanotrophs. J. Cell Sci. 112 (Pt 22): 4143–4150 (1999).

    CAS  PubMed  Google Scholar 

  21. Rupnik, M. et al. Rapid regulated dense-core vesicle exocytosis requires the CAPS protein. Proc. Natl. Acad. Sci. USA 97, 5627–5632 (2000).

    Article  CAS  Google Scholar 

  22. Kreft, M. et al. Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells. Am. J. Physiol. Cell Physiol. 284, C547–C554 (2003).

    Article  CAS  Google Scholar 

  23. Rupnik, M. et al. Distinct role of Rab3A and Rab3B in secretory activity of rat melanotrophs. Am. J. Physiol. Cell Physiol. 292, C98–C105 (2007).

    Article  CAS  Google Scholar 

  24. Coorssen, J.R., Schmitt, H. & Almers, W. Ca2+ triggers massive exocytosis in Chinese hamster ovary cells. EMBO J. 15, 3787–3791 (1996).

    Article  CAS  Google Scholar 

  25. Parsons, T.D., Coorssen, J.R., Horstmann, H. & Almers, W. Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells. Neuron 15, 1085–1096 (1995).

    Article  CAS  Google Scholar 

  26. Sikdar, S.K., Kreft, M. & Zorec, R. Modulation of the unitary exocytic event amplitude by cAMP in rat melanotrophs. J. Physiol. 511 (Pt 3): 851–859 (1998).

    Article  CAS  Google Scholar 

  27. Darios, F. et al. Sphingosine facilitates SNARE complex assembly and activates synaptic vesicle exocytosis. Neuron 62, 683–694 (2009).

    Article  CAS  Google Scholar 

  28. Zupancic, G. et al. The separation of exocytosis from endocytosis in rat melanotroph membrane capacitance records. J. Physiol. 480 (Pt 3): 539–552 (1994).

    Article  CAS  Google Scholar 

  29. Fernandez, J.M., Neher, E. & Gomperts, B.D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. Nature 312, 453–455 (1984).

    Article  CAS  Google Scholar 

  30. Breckenridge, L.J. & Almers, W. Currents through the fusion pore that forms during exocytosis of a secretory vesicle. Nature 328, 814–817 (1987).

    Article  CAS  Google Scholar 

  31. Zimmerberg, J., Curran, M., Cohen, F.S. & Brodwick, M. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc. Natl. Acad. Sci. USA 84, 1585–1589 (1987).

    Article  CAS  Google Scholar 

  32. Alvarez de Toledo, G. & Fernandez, J.M. The events leading to secretory granule fusion. Soc. Gen. Physiol. Ser. 43, 333–344 (1988).

    CAS  PubMed  Google Scholar 

  33. Monck, J.R., Alvarez de Toledo, G. & Fernandez, J.M. Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore. Proc. Natl. Acad. Sci. USA 87, 7804–7808 (1990).

    Article  CAS  Google Scholar 

  34. Spruce, A.E., Breckenridge, L.J., Lee, A.K. & Almers, W. Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle. Neuron 4, 643–654 (1990).

    Article  CAS  Google Scholar 

  35. Oberhauser, A.F. & Fernandez, J.M. Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion. Biophys. J. 69, 451–459 (1995).

    Article  CAS  Google Scholar 

  36. Albillos, A. et al. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389, 509–512 (1997).

    Article  CAS  Google Scholar 

  37. Kreft, M. & Zorec, R. Cell-attached measurements of attofarad capacitance steps in rat melanotrophs. Pflugers Arch. 434, 212–214 (1997).

    Article  CAS  Google Scholar 

  38. Klyachko, V.A. & Jackson, M.B. Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals. Nature 418, 89–92 (2002).

    Article  CAS  Google Scholar 

  39. Vardjan, N., Stenovec, M., Jorgacevski, J., Kreft, M. & Zorec, R. Subnanometer fusion pores in spontaneous exocytosis of peptidergic vesicles. J. Neurosci. 27, 4737–4746 (2007).

    Article  CAS  Google Scholar 

  40. Jorgacevski, J. et al. Hypotonicity and peptide discharge from a single vesicle. Am. J. Physiol. Cell Physiol. 295, C624–C631 (2008).

    Article  CAS  Google Scholar 

  41. Jorgacevski, J. et al. Fusion pore stability of peptidergic vesicles. Mol. Membr. Biol. 27, 65–80 (2010).

    Article  CAS  Google Scholar 

  42. Jorgacevski, J. et al. Munc18-1 tuning of vesicle merger and fusion pore properties. J. Neurosci. 31, 9055–9066 (2011).

    Article  CAS  Google Scholar 

  43. Debus, K. & Lindau, M. Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles. Biophys. J. 78, 2983–2997 (2000).

    Article  CAS  Google Scholar 

  44. Calejo, A.I. et al. Aluminium-induced changes of fusion pore properties attenuate prolactin secretion in rat pituitary lactotrophs. Neuroscience 201, 57–66 (2012).

    Article  CAS  Google Scholar 

  45. Rituper, B., Flasker, A., Gucek, A., Chowdhury, H.H. & Zorec, R. Cholesterol and regulated exocytosis: a requirement for unitary exocytotic events. Cell Calcium 52, 250–258 (2012).

    Article  CAS  Google Scholar 

  46. Tester, M. & Zorec, R. Cytoplasmic calcium stimulates exocytosis in a plant secretory cell. Biophys. J. 63, 864–867 (1992).

    Article  CAS  Google Scholar 

  47. Zorec, R. & Tester, M. Rapid pressure driven exocytosis-endocytosis cycle in a single plant cell. Capacitance measurements in aleurone protoplasts. FEBS Lett. 333, 283–286 (1993).

    Article  CAS  Google Scholar 

  48. Thiel, G., Kreft, M. & Zorec, R. Rhythmic kinetics of single fusion and fission in a plant cell protoplast. Ann. N Y Acad. Sci. 1152, 1–6 (2009).

    Article  CAS  Google Scholar 

  49. Bandmann, V., Kreft, M. & Homann, U. Modes of exocytotic and endocytotic events in tobacco BY-2 protoplasts. Mol. Plant 4, 241–251 (2011).

    Article  CAS  Google Scholar 

  50. Weise, R., Kreft, M., Zorec, R., Homann, U. & Thiel, G. Transient and permanent fusion of vesicles in Zea mays coleoptile protoplasts measured in the cell-attached configuration. J. Membr. Biol. 174, 15–20 (2000).

    Article  CAS  Google Scholar 

  51. Zhang, Q. et al. Fusion-related release of glutamate from astrocytes. J. Biol. Chem. 279, 12724–12733 (2004).

    Article  CAS  Google Scholar 

  52. Pangrsic, T. et al. Exocytotic release of ATP from cultured astrocytes. J. Biol. Chem. 282, 28749–28758 (2007).

    Article  CAS  Google Scholar 

  53. Moser, T. & Beutner, D. Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse. Proc. Natl. Acad. Sci. USA 97, 883–888 (2000).

    Article  CAS  Google Scholar 

  54. Chowdhury, H.H., Kreft, M. & Zorec, R. Rapid insulin-induced exocytosis in white rat adipocytes. Pflugers Arch. 445, 352–356 (2002).

    Article  CAS  Google Scholar 

  55. Graf, J., Rupnik, M., Zupancic, G. & Zorec, R. Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance. Biophys. J. 68, 1359–1363 (1995).

    Article  CAS  Google Scholar 

  56. Kreft, M., Krizaj, D., Grilc, S. & Zorec, R. Properties of exocytotic response in vertebrate photoreceptors. J. Neurophysiol. 90, 218–225 (2003).

    Article  CAS  Google Scholar 

  57. Vandenbeuch, A., Zorec, R. & Kinnamon, S.C. Capacitance measurements of regulated exocytosis in mouse taste cells. J. Neurosci. 30, 14695–14701.

  58. Wightman, R.M. et al. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. USA 88, 10754–10758 (1991).

    Article  CAS  Google Scholar 

  59. Alvarez de Toledo, G., Fernández-Chacón, R. & Fernández, J.M. Release of secretory products during transient vesicle fusion. Nature 363, 554–558 (1993).

    Article  CAS  Google Scholar 

  60. Betz, W.J., Mao, F. & Bewick, G.S. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12, 363–375 (1992).

    Article  CAS  Google Scholar 

  61. Stenovec, M., Poberaj, I., Kreft, M. & Zorec, R. Concentration-dependent staining of lactotroph vesicles by FM 4-64. Biophys. J. 88, 2607–2613 (2005).

    Article  CAS  Google Scholar 

  62. Miesenböck, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  Google Scholar 

  63. Malarkey, E.B. & Parpura, V. Temporal characteristics of vesicular fusion in astrocytes: examination of synaptobrevin 2-laden vesicles at single vesicle resolution. J. Physiol. 589, 4271–4300 (2011).

    Article  CAS  Google Scholar 

  64. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    Article  CAS  Google Scholar 

  65. Speidel, D. et al. CAPS1 regulates catecholamine loading of large dense-core vesicles. Neuron 46, 75–88 (2005).

    Article  CAS  Google Scholar 

  66. Neef, A., Heinemann, C. & Moser, T. Measurements of membrane patch capacitance using a software-based lock-in system. Pflugers Arch. 454, 335–344 (2007).

    Article  CAS  Google Scholar 

  67. Lindau, M. & Neher, E. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch. 411, 137–146 (1988).

    Article  CAS  Google Scholar 

  68. Gillis, K.D. Techniques for membrane capacitance measurements. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 155–198 (Plenum Press, 1995).

  69. Rae, J., Cooper, K., Gates, P. & Watsky, M. Low access resistance perforated patch recordings using amphotericin B. J. Neurosci. Methods 37, 15–26 (1991).

    Article  CAS  Google Scholar 

  70. Horn, R. & Marty, A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145–159 (1988).

    Article  CAS  Google Scholar 

  71. Lippiat, J.D. Whole-cell recording using the perforated patch clamp technique. Methods Mol. Biol. 491, 141–149 (2008).

    Article  CAS  Google Scholar 

  72. Horrigan, F.T. & Bookman, R.J. Releasable pools and the kinetics of exocytosis in adrenal chromaffin cells. Neuron 13, 1119–1129 (1994).

    Article  CAS  Google Scholar 

  73. Sakmann, B. & Neher, E. Single-Channel Recording (Plenum Press, 1995).

  74. Heinemann, S. Guide to data acquisition and analysis. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 53–91 (Plenum Press, 1995).

  75. Poberaj, I., Rupnik, M., Kreft, M., Sikdar, S.K. & Zorec, R. Modeling excess retrieval in rat melanotroph membrane capacitance records. Biophys. J. 82, 226–232 (2002).

    Article  CAS  Google Scholar 

  76. Ben-Tabou, S., Keller, E. & Nussinovitch, I. Mechanosensitivity of voltage-gated calcium currents in rat anterior pituitary cells. J. Physiol. 476, 29–39 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Mitchner, N.A., Garlick, C. & Ben-Jonathan, N. Cellular distribution and gene regulation of estrogen receptors-α and -β in the rat pituitary gland. Endocrinology 139, 3976–3983 (1998).

    Article  CAS  Google Scholar 

  78. Rupnik, M. & Zorec, R. Intracellular Cl− modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins. Pflugers Arch. 431, 76–83 (1995).

    Article  CAS  Google Scholar 

  79. Lindau, M. Time-resolved capacitance measurements: monitoring exocytosis in single cells. Q. Rev. Biophys. 24, 75–101 (1991).

    Article  CAS  Google Scholar 

  80. Rituper, B. et al. Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells. Biochim. Biophys. Acta 1831, 1228–1238 (2013).

    Article  CAS  Google Scholar 

  81. Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717–766 (2000).

    Article  CAS  Google Scholar 

  82. Harper, C.B. et al. Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J. Biol. Chem. 286, 35966–35976 (2011).

    Article  CAS  Google Scholar 

  83. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006).

    Article  CAS  Google Scholar 

  84. Pusch, M. & Neher, E. Rates of diffusional exchange between small cells and a measuring patch pipette. Pflugers Arch. 411, 204–211 (1988).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, Ljubljana, Slovenia

    Boštjan Rituper, Alenka Guček, Jernej Jorgačevski, Ajda Flašker, Marko Kreft & Robert Zorec

  2. Celica Biomedical Center, Ljubljana, Slovenia

    Jernej Jorgačevski, Marko Kreft & Robert Zorec

  3. Biotechnical Faculty, Ljubljana, Slovenia

    Marko Kreft

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  1. Boštjan Rituper
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Contributions

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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