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Analysis of Electrophysiological Properties and Responses of Neutrophils

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Book cover Neutrophil Methods and Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 412))

Abstract

The past decade has seen increasing use of the patch clamp technique on neutrophils and eosinophils. The main goal of these electrophysiological studies has been to elucidate the mechanisms underlying the phagocyte respiratory burst. NADPH oxidase activity, which defines the respiratory burst in granulocytes, is electrogenic because electrons from NADPH are transported across the cell membrane, where they reduce oxygen to form superoxide anion (O2 −). This passage of electrons comprises an electrical current that would rapidly depolarize the membrane if the charge movement were not balanced by proton efflux. The patch clamp technique enables simultaneous recording of NADPH oxidase-generated electron current and H+ flux through the closely related H+ channel. Increasing evidence suggests that other ion channels may play crucial roles in degranulation, phagocytosis, and chemotaxis, highlighting the importance of electrophysiological studies to advance knowledge of granulocyte function. Several configurations of the patch clamp technique exist. Each has advantages and limitations that are discussed here. Meaningful measurements of ion channels cannot be achieved without an understanding of their fundamental properties. We describe the types of measurements that are necessary to characterize a particular ion channel.

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References

  1. Hodgkin, A. L. and Huxley, A. F. (1952) Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. 116, 449–472.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  3. von Tscharner, V., Prod’hom, B., Baggiolini, M., and Reuter, H. (1986) Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration. Nature 324, 369–372.

    Article  Google Scholar 

  4. DeCoursey, T. E. and Cherny, V. V. (1993) Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils. Biophys. J. 65, 1590–1598.

    Article  PubMed  CAS  Google Scholar 

  5. Stoddard, J. S., Steinbach, J. H., and Simchowitz, L. (1993) Whole cell Cl− currents in human neutrophils induced by cell swelling. Am. J. Physiol. 265, C156–C165.

    PubMed  CAS  Google Scholar 

  6. Gordienko, D. V., Tare, M., Parveen, S., Fenech, C. J., Robinson, C., and Bolton, T. B. (1996) Voltage-activated proton current in eosinophils from human blood. J. Physiol. 496, 299–316.

    PubMed  CAS  Google Scholar 

  7. Schrenzel, J., Serrander, L., Bánfi, B., et al. (1998) Electron currents generated by the human phagocyte NADPH oxidase. Nature 392, 734–737.

    Article  PubMed  CAS  Google Scholar 

  8. Tare, M., Prestwich, S. A., Gordienko, S., et al. (1998) Inwardly rectifying whole cell potassium current in human blood eosinophils. J. Physiol. 506, 303–318.

    Article  PubMed  CAS  Google Scholar 

  9. Babior, B. M. (1999) NADPH oxidase: an update. Blood 93, 1464–1476.

    PubMed  CAS  Google Scholar 

  10. DeCoursey, T. E., Cherny, V. V., Zhou, W., and Thomas, L. L. (2000) Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils. Proc. Natl. Acad. Sci. USA 97, 6885–6889.

    Article  PubMed  CAS  Google Scholar 

  11. Petheö, G. L., Maturana, A., Spat, A., and Demaurex, N. (2003) Interactions between electron and proton currents in excised patches from human eosinophils. J. Gen. Physiol. 122, 713–726.

    Article  PubMed  Google Scholar 

  12. DeCoursey, T. E., Morgan, D., and Cherny, V. V. (2003) The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels. Nature 422, 531–534.

    Article  PubMed  CAS  Google Scholar 

  13. Morgan, D., Cherny, V. V., Murphy, R., Xu, W., Thomas, L. L., and DeCoursey, T. E. (2003) Temperature dependence of NADPH oxidase in human eosinophils. J. Physiol. 550, 447–458.

    Article  PubMed  CAS  Google Scholar 

  14. Petheö, G. L. and Demaurex, N. (2005) Voltage-and NADPH-dependence of electron currents generated by the phagocytic NADPH oxidase. Biochem. J. 388, 485–491.

    Article  PubMed  Google Scholar 

  15. Morgan, D., Cherny, V. V., Murphy, R., Katz, B., and DeCoursey, T. E. (2005) pH dependence of PMA activated electron current in human neutrophils. J. Physiol. 569, 419–431.

    Article  PubMed  CAS  Google Scholar 

  16. Menegazzi, R., Busetto, S., Dri, P., Cramer, R., and Patriarca, P. (1996) Chloride ion efflux regulates adherence, spreading and respiratory burst of neutrophils stimulated by tumor necrosis factor-α (TNF) on biological surfaces. J. Cell Biol. 135, 511–522.

    Article  PubMed  CAS  Google Scholar 

  17. Moreland, J. G., Davis, A. P. Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration. J. Biol. Chem. 281, 12,277–12,288.

    Article  PubMed  CAS  Google Scholar 

  18. Cherny, V. V., Henderson, L. M., and DeCoursey, T. E. (1997) Proton and chloride currents in Chinese hamster ovary cells. Membr. Cell Biol. 11, 337–340.

    PubMed  CAS  Google Scholar 

  19. Demaurex, N., Grinstein, S., Jaconi, M., Schlegel, W., Lew, D. P., and Krause, K.-H. (1993) Proton currents in human granulocytes: regulation by membrane potential and intracellular pH. J. Physiol. 466, 329–344.

    PubMed  CAS  Google Scholar 

  20. DeCoursey, T. E. and Cherny V. V. (1994) Voltage-activated hydrogen ion currents. J. Membr. Biol. 141, 203–223.

    PubMed  CAS  Google Scholar 

  21. DeCoursey, T. E. and Cherny, V. V. (1996) Effects of buffer concentration on voltage-gated H+ currents: does diffusion limit the conductance? Biophys. J. 71, 182–193.

    Article  PubMed  CAS  Google Scholar 

  22. Gordienko, D. V., Tare, M., Parveen, S., Fenech, C. J., Robinson, C., and Bolton, T. B. (1996) Voltage-activated proton current in eosinophils from human blood. J. Physiol. 496, 299–316.

    PubMed  CAS  Google Scholar 

  23. Schrenzel, J., Lew, D. P., and Krause, K.-H. (1996) Proton currents in human eosinophils. Am. J. Physiol. 271, C1861–C1871.

    PubMed  CAS  Google Scholar 

  24. Cherny, V. V. and DeCoursey, T. E. (1999) pH-dependent inhibition of voltagegated H+ currents in rat alveolar epithelial cells by Zn2+ and other divalent cations. J. Gen. Physiol. 114, 819–838.

    Article  PubMed  CAS  Google Scholar 

  25. Femling, J. K., Cherny, V. V., Morgan, D., et al. (2006) The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels. J. Gen. Physiol. 127, 659–672.

    Article  PubMed  CAS  Google Scholar 

  26. Grinstein, S., Romanek, R., and Rotstein, O. D. (1994) Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated patch configurations. Am. J. Physiol. 267, C1152–C1159.

    PubMed  CAS  Google Scholar 

  27. DeCoursey, T. E. and Cherny, V. V. (1998) Temperature dependence of voltagegated H+ currents in human neutrophils, rat alveolar epithelial cells, and mammalian phagocytes. J. Gen. Physiol. 112, 503–522.

    Article  PubMed  CAS  Google Scholar 

  28. Chabala, L. D., Sheridan, R. E., Hodge, D. C., Power, J. N., and Walsh, M. P. (1985) A microscope stage temperature controller for the study of whole-cell or single-channel currents. Pflügers Arch. 404, 374–377.

    Article  PubMed  CAS  Google Scholar 

  29. Rae, J. L. and Levis, R. A. (1984) Patch voltage clamp of lens epithelial cells: theory and practice. Mol. Physiol. 6, 115–162.

    CAS  Google Scholar 

  30. Fenwick, E. M., Marty, A., and Neher, E. (1982) A patch clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physiol. 331, 577–597.

    PubMed  CAS  Google Scholar 

  31. Lindau, M. and Fernandez, J. M. (1986) IgE-mediated degranulation of mast cells does not require opening of ion channels. Nature 319, 150–153.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  34. Fan, J. S. and Palade, P. (1998) Perforated patch recording with β-escin. Pflügers Arch. 436, 1021–1023.

    Article  PubMed  CAS  Google Scholar 

  35. Falke, L. C., Gillis, K. D., Pressel, D. M., and Misler, S. (1989) ‘Perforated patch recording’ allows long-term monitoring of metabolite-induced electrical activity and voltage-dependent Ca2+ currents in pancreatic islet B cells. FEBS Lett. 251, 167–172.

    Article  PubMed  CAS  Google Scholar 

  36. Chung, I. and Schlichter, L. C. (1993) Criteria for perforated-patch recordings: ion currents versus dye permeation in human T lymphocytes. Pflügers Arch. 424, 511–515.

    Article  PubMed  CAS  Google Scholar 

  37. Strauss, U., Herbrik, M., Mix, E., Schubert, R., and Rolfs, A. (2001) Whole-cell patch-clamp: true perforated or spontaneous conventional recordings? Pflügers Arch. 442, 634–638.

    Article  PubMed  CAS  Google Scholar 

  38. Bánfi, B., Schrenzel, J., Nüsse, O., et al. (1999) A novel H+ conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease. J. Exp. Med. 19, 183–194.

    Article  Google Scholar 

  39. Robertson, A. K., Cross, A. R., Jones, O. T. G., and Andrew, P. W. (1990) The use of diphenylene iodonium, an inhibitor of NADPH oxidase, to investigate the antimicrobial action of human monocyte derived macrophages. J. Immunol. Methods 133, 175–179.

    Article  PubMed  CAS  Google Scholar 

  40. DeCoursey, T. E. and Cherny, V. V. (1994) Na+-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils. J. Gen. Physiol. 103, 755–785.

    Article  PubMed  CAS  Google Scholar 

  41. DeCoursey, T. E. (1991) Hydrogen ion currents in rat alveolar epithelial cells. Biophys. J. 60, 1243–1253.

    Article  PubMed  CAS  Google Scholar 

  42. Byerly, L., Meech, R., and Moody, W. Jr. (1984) Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymnaea stagnalis. J. Physiol. 351, 199–216.

    PubMed  CAS  Google Scholar 

  43. Humez, S., Fournier, F., and Guilbault, P. (1995) A voltage-dependent and pH-sensitive proton current in Rana esculenta oocytes. J. Membr. Biol. 147, 207–215.

    PubMed  CAS  Google Scholar 

  44. Hille, B. (2001) Ion Channels of Excitable Membranes, 3rd edition. Sinauer, MA.

    Google Scholar 

  45. Cherny, V. V., Murphy, R., Sokolov, V., Levis, R. A., and DeCoursey, T. E. (2003) Properties of single voltage-gated proton channels in human eosinophils estimated by noise analysis and direct measurement. J. Gen. Physiol. 121, 615–628.

    Article  PubMed  CAS  Google Scholar 

  46. Neher, E. and Stevens, C. F. (1977) Conductance fluctuations and ionic pores in membranes. Annu. Rev. Biophys. Bioeng. 6, 345–381.

    Article  PubMed  CAS  Google Scholar 

  47. Demaurex, N., Monod, A., Lew, D. P., and Krause, K.-H. (1994) Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils. Biochem. J. 297, 595–601.

    PubMed  CAS  Google Scholar 

  48. Zweifach, A. and Lewis, R. S. (1993) Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc. Natl. Acad. Sci. USA 90, 6295–6299.

    Article  PubMed  CAS  Google Scholar 

  49. Cross, A. R., Higson, F. K., Jones, O. T. G., Harper, A. M., and Segal, A. W. (1982) The enzymic reduction and kinetics of oxidation of cytochrome b −245 of neutrophils. Biochem. J. 204, 479–485.

    PubMed  CAS  Google Scholar 

  50. Murphy, R. and DeCoursey, T. E. (2006) Charge compensation during the phagocyte respiratory burst. Biochim. Biophys. Acta 1757, 996–1011.

    Article  PubMed  CAS  Google Scholar 

  51. Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem. J. 246, 325–329.

    PubMed  CAS  Google Scholar 

  52. Geiszt, M., Kapus, A., Nemet, K., Farkas, L., and Ligeti, E. (1997) Regulation of capacitative Ca2+ influx in human neutrophil granulocytes. Alterations in chronic granulomatous disease. J. Biol. Chem. 272, 26,471–26,478.

    Article  PubMed  CAS  Google Scholar 

  53. Jankowski, A. and Grinstein, S. (1999) A noninvasive fluorometric procedure for measurement of membrane potential. J. Biol. Chem. 274, 26,098–26,104.

    Article  PubMed  CAS  Google Scholar 

  54. Bankers-Fulbright, J. L., Gleich, G. J., Kephart, G. M., Kita, H., and O’Grady, S. M. (2003) Regulation of eosinophil membrane depolarization during NADPH oxidase activation. J. Cell Sci. 116, 3221–3226.

    Article  PubMed  CAS  Google Scholar 

  55. Rada, B. K., Geiszt, M., Káldi, K., Tímár, C., and Ligeti, E. (2004) Dual role of phagocytic NADPH oxidase in bacterial killing. Blood 104, 2947–2953.

    Article  PubMed  CAS  Google Scholar 

  56. Demaurex, N. and Petheõ, G. L. (2005) Electron and proton transport by NADPH oxidases. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360, 2315–2325.

    Article  PubMed  CAS  Google Scholar 

  57. Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem. J. 886, 425–433.

    Google Scholar 

  58. DeCoursey, T. E. (2004) During the respiratory burst, do phagocytes need proton channels or potassium channels or both? Science’s STKE 2004, pe21 (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/233/pe21?ijkey=lPoLtn7p09qzw&keytype=ref&siteid=sigtrans).

    Google Scholar 

  59. Rae, J. L. and Levis, R. A. (1984) Patch voltage clamp of lens epithelial cells: theory and practice. Mol. Physiol. 6, 115–162.

    CAS  Google Scholar 

  60. Sigworth, F. J. (1995) Electronic design of the patch clamp, in Single-Channel Recording, 2nd edition (Sakmann, B., and Neher, E. eds.), Plenum, NY, pp. 95–127.

    Google Scholar 

  61. Byerly, L. and Moody, W. J. (1986) Membrane currents of internally perfused neurons of the snail, Lymnaea stagnalis, at low intracellular pH. J. Physiol. 376, 477–491.

    PubMed  CAS  Google Scholar 

  62. Kapus, A., Romanek, R., Qu, A. Y., Rotstein, O. D., and Grinstein, S. (1993) A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages. J. Gen. Physiol. 102, 729–760.

    Article  PubMed  CAS  Google Scholar 

  63. Levis, R. and Rae, J. L. (1992) Constructing a patch clamp setup. Meth. Enzymol. 207, 14–66.

    Article  PubMed  CAS  Google Scholar 

  64. Levis, R. A. and Rae, J. L. (1993) The use of quartz patch pipettes for low noise single channel recording. Biophys. J. 65, 1666–1677.

    Article  PubMed  CAS  Google Scholar 

  65. Cota, G. and Armstrong, C. M. (1988) Potassium channel “inactivation” induced by soft-glass pipettes. Biophys. J. 55, 107–109.

    Article  Google Scholar 

  66. Rojas, L. and Zuazaga, C. (1988) Influence of the patch pipette glass on single acetylcholine channels recorded from Xenopus myocytes. Neurosci. Lett. 88, 39–44.

    Article  PubMed  CAS  Google Scholar 

  67. Neher, E. (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207, 123–131.

    Article  PubMed  CAS  Google Scholar 

  68. Ng, B. and Barry, P. H. (1995) The measurement of ionic conductivities and mobilities of certain less common organic ions needed for junction potential corrections in electrophysiology. J. Neurosci. Methods 56, 37–41.

    Article  PubMed  CAS  Google Scholar 

  69. Miller, C., Moczydlowski, E., Latorre, R., and Phillips, M. (1985) Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature 313, 316–318.

    Article  PubMed  CAS  Google Scholar 

  70. Galvez, A., Gimenez-Gallego, G., Reuben, J. P., et al. (1990) Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J. Biol. Chem. 265, 11,083–11,090.

    PubMed  CAS  Google Scholar 

  71. Hermann, A. and Erxleben, C. (1987) Charybdotoxin selectively blocks small Caactivated K channels in Aplysia neurons. J. Gen. Physiol. 90, 27–47.

    Article  PubMed  CAS  Google Scholar 

  72. Sands, S. B., Lewis, R. S., and Cahalan, M. D. (1989) Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes. J. Gen. Physiol. 93, 1061–1074.

    Article  PubMed  CAS  Google Scholar 

  73. Cherny, V. V., Markin, V. S., and DeCoursey, T. E. (1995) The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. J. Gen. Physiol. 105, 861–896.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL

    Deri Morgan & Thomas E. DeCoursey

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  1. Deri Morgan
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  2. Thomas E. DeCoursey
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Editors and Affiliations

  1. Deparment of Veterinary Molecular Biology, Montana State University, Bozeman, MT

    Mark T. Quinn

  2. Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT

    Frank R. DeLeo

  3. Departments of Immunology and Cell Biology, The Scripps Institute, La Jolla, CA

    Gary M. Bokoch

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Morgan, D., DeCoursey, T.E. (2007). Analysis of Electrophysiological Properties and Responses of Neutrophils. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_11

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  • DOI: https://doi.org/10.1007/978-1-59745-467-4_11

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-788-4

  • Online ISBN: 978-1-59745-467-4

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