Measurements of Exocytosis by Capacitance Recordings and Calcium Uncaging in Mouse Adrenal Chromaffin Cells

  • Sébastien Houy
  • Joana S. Martins
  • Ralf Mohrmann
  • Jakob Balslev SørensenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2233)


Fusion of vesicles with the plasma membrane and liberation of their contents is a multistep process involving several proteins. Correctly assigning the role of specific proteins and reactions in this cascade requires a measurement method with high temporal resolution. Patch-clamp recordings of cell membrane capacitance in combination with calcium measurements, calcium uncaging, and carbon-fiber amperometry allow for the accurate determination of vesicle pool sizes, their fusion kinetics, and their secreted oxidizable content. Here, we will describe this method in a model system for neurosecretion, the adrenal chromaffin cells, which secrete adrenaline.

Key words

Neurosecretion Exocytosis Vesicles Chromaffin cells Capacitance measurement Carbon-fiber amperometry Patch-clamp Electrophysiology Calcium measurements 


  1. 1.
    Sorensen JB (2004) Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles. Pflugers Archiv 448:347–362PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Verhage M, Sorensen JB (2008) Vesicle docking in regulated exocytosis. Traffic 9:1414–1424PubMedCrossRefGoogle Scholar
  3. 3.
    Neher E (2018) Neurosecretion: what can we learn from chromaffin cells. Pflugers Archiv 470:7–11PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Rettig J, Neher E (2002) Emerging roles of presynaptic proteins in Ca++-triggered exocytosis. Science 298:781–785PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Marengo FD, Cardenas AM (2018) How does the stimulus define exocytosis in adrenal chromaffin cells? Pflugers Arch 470:155–167PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Dhara M, Mohrmann R, Bruns D (2018) v-SNARE function in chromaffin cells. Pflugers Arch 470:169–180PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Stevens DR, Schirra C, Becherer U, Rettig J (2011) Vesicle pools: lessons from adrenal chromaffin cells. Front Synaptic Neurosci 3:2PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Bader MF, Holz RW, Kumakura K, Vitale N (2002) Exocytosis: the chromaffin cell as a model system. Ann N Y Acad Sci 971:178–183PubMedCrossRefGoogle Scholar
  9. 9.
    Steyer JA, Horstmann H, Almers W (1997) Transport, docking and exocytosis of single secretory granules in live chromaffin cells. Nature 388:474–478PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Sorensen JB, Nagy G, Varoqueaux F, Nehring RB, Brose N, Wilson MC, Neher E (2003) Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23. Cell 114:75–86PubMedCrossRefGoogle Scholar
  11. 11.
    Borisovska M, Zhao Y, Tsytsyura Y, Glyvuk N, Takamori S, Matti U, Rettig J, Sudhof T, Bruns D (2005) v-SNAREs control exocytosis of vesicles from priming to fusion. EMBO J 24:2114–2126PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Voets T, Moser T, Lund PE, Chow RH, Geppert M, Sudhof TC, Neher E (2001) Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I. Proc Natl Acad Sci U S A 98:11680–11685PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Voets T, Toonen RF, Brian EC, de Wit H, Moser T, Rettig J, Sudhof TC, Neher E, Verhage M (2001) Munc18-1 promotes large dense-core vesicle docking. Neuron 31:581–591PubMedCrossRefGoogle Scholar
  14. 14.
    Schonn JS, Maximov A, Lao Y, Sudhof TC, Sorensen JB (2008) Synaptotagmin-1 and -7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells. Proc Natl Acad Sci U S A 105:3998–4003PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Liu Y, Schirra C, Stevens DR, Matti U, Speidel D, Hof D, Bruns D, Brose N, Rettig J (2008) CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles. J Neurosci 28:5594–5601PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Speidel D, Bruederle CE, Enk C, Voets T, Varoqueaux F, Reim K, Becherer U, Fornai F, Ruggieri S, Holighaus Y, Weihe E, Bruns D, Brose N, Rettig J (2005) CAPS1 regulates catecholamine loading of large dense-core vesicles. Neuron 46:75–88PubMedCrossRefGoogle Scholar
  17. 17.
    Man KN, Imig C, Walter AM, Pinheiro PS, Stevens DR, Rettig J, Sorensen JB, Cooper BH, Brose N, Wojcik SM (2015) Identification of a Munc13-sensitive step in chromaffin cell large dense-core vesicle exocytosis. eLife 4:e10635PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Ashery U, Varoqueaux F, Voets T, Betz A, Thakur P, Koch H, Neher E, Brose N, Rettig J (2000) Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells. EMBO J 19:3586–3596PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Cai H, Reim K, Varoqueaux F, Tapechum S, Hill K, Sorensen JB, Brose N, Chow RH (2008) Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming. Proc Natl Acad Sci U S A 105:19538–19543PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Vitale ML, Seward EP, Trifaro JM (1995) Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis. Neuron 14:353–363PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Houy S, Groffen AJ, Ziomkiewicz I, Verhage M, Pinheiro PS, Sorensen JB (2017) Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs. eLife 6:e27000PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Sorensen JB, Wiederhold K, Muller EM, Milosevic I, Nagy G, de Groot BL, Grubmuller H, Fasshauer D (2006) Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles. EMBO J 25:955–966PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Mohrmann R, de Wit H, Verhage M, Neher E, Sorensen JB (2010) Fast vesicle fusion in living cells requires at least three SNARE complexes. Science 330:502–505PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Shaaban A, Dhara M, Frisch W, Harb A, Shaib AH, Becherer U, Bruns D, Mohrmann R (2019) The SNAP-25 linker supports fusion intermediates by local lipid interactions. eLife 8:e41720PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Makke M, Mantero Martinez M, Gaya S, Schwarz Y, Frisch W, Silva-Bermudez L, Jung M, Mohrmann R, Dhara M, Bruns D (2018) A mechanism for exocytotic arrest by the Complexin C-terminus. eLife 7:e38981PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Mohrmann R, de Wit H, Connell E, Pinheiro PS, Leese C, Bruns D, Davletov B, Verhage M, Sorensen JB (2013) Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering. J Neurosci 33:14417–14430PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Angleson JK, Betz WJ (1997) Monitoring secretion in real time: capacitance, amperometry and fluorescence compared. Trends Neurosci 20:281–287PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Khvotchev M, Kavalali ET (2008) Pharmacology of neurotransmitter release: measuring exocytosis. Handb Exp Pharmacol 184:23–43CrossRefGoogle Scholar
  29. 29.
    Neher E, Marty A (1982) Discrete changes of cell-membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci U S A 79:6712–6716PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Borges R, Camacho M, Gillis KD (2008) Measuring secretion in chromaffin cells using electrophysiological and electrochemical methods. Acta Physiol 192:173–184CrossRefGoogle Scholar
  31. 31.
    Lindau M, Neher E (1988) Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch 411:137–146PubMedCrossRefGoogle Scholar
  32. 32.
    Gillis KD (1995) Techniques for membrane capacitance measurements. In: Sakmann B, Neher E (eds) Single-channel recording, 2nd edn. Plenum Press, New York, pp 155–198CrossRefGoogle Scholar
  33. 33.
    Segev A, Garcia-Oscos F, Kourrich S (2016) Whole-cell patch-clamp recordings in brain slices. J Vis Exp 112:54024Google Scholar
  34. 34.
    Conforti L (2012). Chapter 20: patch-clamp technique. In: Sperelakis N (ed) Cell physiology source book, 4th edn. Academic, CambridgeGoogle Scholar
  35. 35.
    Heinemann C, Chow RH, Neher E, Zucker RS (1994) Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+. Biophys J 67:2546–2557PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Ellis-Davies GC, Kaplan JH (1994) Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci U S A 91:187–191PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Voets T (2000) Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices. Neuron 28:537–545PubMedCrossRefGoogle Scholar
  38. 38.
    Sorensen JB, Matti U, Wei SH, Nehring RB, Voets T, Ashery U, Binz T, Neher E, Rettig J (2002) The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis. Proc Natl Acad Sci U S A 99:1627–1632PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Wightman RM, Jankowski JA, Kennedy RT, Kawagoe KT, Schroeder TJ, Leszczyszyn DJ, Near JA, Diliberto EJ Jr, Viveros OH (1991) Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc Natl Acad Sci U S A 88:10754–10758PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Chow RH, von Ruden L, Neher E (1992) Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356:60–63PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Fathali H, Cans AS (2018) Amperometry methods for monitoring vesicular quantal size and regulation of exocytosis release. Pflugers Archiv 470:125–134PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Mosharov EV, Sulzer D (2005) Analysis of exocytotic events recorded by amperometry. Nat Methods 2:651–658PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Bruns D (2004) Detection of transmitter release with carbon fiber electrodes. Methods 33:312–321PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Chow RH, Rüden L (1995) Electrochemical detection of secretion from single cells. In: Sakmann B, Neher E (eds) Single-channel recording, 2nd edn. Plenum Press, New YorkGoogle Scholar
  45. 45.
    Penner R (1995) A practical guide to patch clamping. In: Sakmann B, Neher E (eds) Single-channel recording, 2nd edn. Plenum Press, New YorkGoogle Scholar
  46. 46.
    Lindau M, Neher E (1988) Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Archiv 411:137–146PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Chen P, Gillis KD (2000) The noise of membrane capacitance measurements in the whole-cell recording configuration. Biophys J 79:2162–2170PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Horrigan FT, Bookman RJ (1994) Releasable pools and the kinetics of exocytosis in adrenal chromaffin cells. Neuron 13:1119–1129PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Voets T, Neher E, Moser T (1999) Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices. Neuron 23:607–615PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Yang Y, Gillis KD (2004) A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells. J Gen Physiol 124:641–651PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Ellis-Davies GC (2008) Neurobiology with caged calcium. Chem Rev 108:1603–1613PubMedCrossRefGoogle Scholar
  52. 52.
    Xu T, Binz T, Niemann H, Neher E (1998) Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci 1:192–200PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Sébastien Houy
    • 1
  • Joana S. Martins
    • 1
  • Ralf Mohrmann
    • 2
  • Jakob Balslev Sørensen
    • 1
    Email author
  1. 1.Department of NeuroscienceUniversity of CopenhagenCopenhagen NDenmark
  2. 2.Institute for PhysiologyOtto-von-Guericke UniversityMagdeburgGermany

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