Advertisement

Perforated Whole-Cell Patch-Clamp Technique: A User’s Guide

  • Hitoshi Ishibashi
  • Andrew J. Moorhouse
  • Junichi Nabekura
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The patch-clamp technique has revolutionized the study of membrane physiology, enabling unprecedented resolution in recording cellular electrical responses and underlying mechanisms. The perforated-patch variant of whole-cell patch-clamp recording was developed to overcome the dialysis of cytoplasmic constituents that occurs with traditional whole-cell recording. With perforated-patch recordings, perforants, such as the antibiotics nystatin and gramicidin, are included in the pipette solution and form small pores in the membrane attached to the patch pipette. These pores allow certain monovalent ions to permeate, enabling electrical access to the cell interior, but prevent the dialysis of larger molecules and other ions. In this review we give a brief overview of the key features of some of the perforants, present some practical approaches to the use of the perforated patch-clamp mode of whole-cell (PPWC) recordings, and give some typical examples of neuronal responses obtained with the PPWC recording that highlight its utility as compared to the traditional whole-cell patch recording configuration.

Keywords

Pipette Solution Patch Pipette Access Resistance Polyene Antibiotic Cellular Excitability 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Neher E, Sakmann B (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799–802PubMedCrossRefGoogle Scholar
  2. 2.
    Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches. Pflugers Arch 391:85–100PubMedCrossRefGoogle Scholar
  3. 3.
    Lindau M, Fernandez M (1986) IgE-mediated degranulation of mast cells does not require opening of ion channels. Nature 319:150–153PubMedCrossRefGoogle Scholar
  4. 4.
    Horn R, Marty A (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92:145–159PubMedCrossRefGoogle Scholar
  5. 5.
    Rhee JS, Ebihara S, Akaike N (1994) Gramicidin perforated patch-clamp technique reveals glycine-gated outward chloride current in dissociated nucleus solitarii neurons of rat. J Neurophysiol 72:1103–1108PubMedGoogle Scholar
  6. 6.
    Ebihara S, Shirato K, Harata N, Akaike N (1995) Gramicidin-perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride. J Physiol 484:77–86PubMedGoogle Scholar
  7. 7.
    Akaike N, Harata N (1994) Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. Jpn J Physiol 44:433–473PubMedCrossRefGoogle Scholar
  8. 8.
    Levitan ES, Kramer RH (1990) Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration. Nature 348:545–547PubMedCrossRefGoogle Scholar
  9. 9.
    Korn SJ, Horn R (1989) Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording. J Gen Physiol 94:789–812PubMedCrossRefGoogle Scholar
  10. 10.
    Marsh SJ, Trouslard J, Leaney JL, Brown DA (1995) Synergistic regulation of a neuronal chloride current by intracellular calcium and muscarinic receptor activation: a role for protein kinase C. Neuron 15:729–737PubMedCrossRefGoogle Scholar
  11. 11.
    Rae J, Cooper K, Gates P, Watsky M (1991) Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37:15–26PubMedCrossRefGoogle Scholar
  12. 12.
    Reichling DB, Kyrozis A, Wang J, MacDermott AB (1994) Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons. J Physiol 476:411–421PubMedGoogle Scholar
  13. 13.
    Kyrozis A, Reichling DB (1995) Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride. J Neurosci Methods 57:27–35PubMedCrossRefGoogle Scholar
  14. 14.
    Fan JS, Palade P (1998) Perforated patch recording with β-escin. Pflugers Arch 436:1021–1023PubMedCrossRefGoogle Scholar
  15. 15.
    Sarantopoulos C, McCallum JB, Kwok WM, Hogan Q (2004) β-escin diminishes voltage-gated calcium current rundown in perforated patch-clamp recordings from rat primary afferent neurons. J Neurosci Methods 139:61–68PubMedCrossRefGoogle Scholar
  16. 16.
    Myers VB, Haydon DA (1972) Ion transfer across lipid membranes in the presence of gramicidin A. II. The ion selectivity. Biochim Biophys Acta 274:313–322PubMedCrossRefGoogle Scholar
  17. 17.
    Kleinberg ME, Finkelstein A (1984) Single-length and double length channels formed by nystatin in lipid bilayer membranes. J Membr Biol 80:257–269PubMedCrossRefGoogle Scholar
  18. 18.
    Marty A, Finkelstein A (1975) Pores formed in lipid bilayer membranes by nystatin; Differences in its one-sided and two-sided action. J Gen Physiol 65:515–526PubMedCrossRefGoogle Scholar
  19. 19.
    Tajima Y, Ono K, Akaike N (1996) Perforated patch-clamp recording in cardiac myocytes using cation-selective ionophore gramicidin. Am J Physiol 271:C524–C532PubMedGoogle Scholar
  20. 20.
    Ermishkin LN, Kasumov KM, Potzeluyev VM (1976) Single ionic channels induced in lipid bilayers by polyene antibiotics amphotericin B and nystatine. Nature 262:698–699PubMedCrossRefGoogle Scholar
  21. 21.
    Hladky SB, Haydon DA (1972) Ion transfer across lipid membranes in the presence of gramicidin A. I. Studies of the unit conductance channel. Biochim Biophys Acta 274:294–312PubMedCrossRefGoogle Scholar
  22. 22.
    Holz R, Finkelstein A (1970) The water and nonelectrolyte permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B. J Gen Physiol 56:125–145PubMedCrossRefGoogle Scholar
  23. 23.
    Horn R (1991) Diffusion of nystatin in plasma membrane is inhibited by a glass-membrane seal. Biophys J 60:329–333PubMedCrossRefGoogle Scholar
  24. 24.
    Farrant M, Kaila K (2007) The cellular, molecular and ionic basis of GABAA receptor signaling. Prog Brain Res 160:59–87PubMedCrossRefGoogle Scholar
  25. 25.
    Lenz RA, Pitler TA, Alger B (1997) High intracellular Cl concentrations depress G-protein-modulated ionic conductances. J Neurosci 17:6133–6141PubMedGoogle Scholar
  26. 26.
    Beato M (2008) (2008) The time course of transmitter at glycinergic synapses onto motoneurons. J Neurosci 28:7412–7425PubMedCrossRefGoogle Scholar
  27. 27.
    Yawo H, Chuhma N (1993) An improved method for perforated patch recordings using nystatin-fluorescein mixture. Jpn J Physiol 43:267–273PubMedCrossRefGoogle Scholar
  28. 28.
    Endo K, Yawo H (2000) μ-Opioid receptor inhibits N-type Ca2+ channels in the calyx presynaptic terminal of the embryonic chick ciliary ganglion. J Physiol 524:769–781PubMedCrossRefGoogle Scholar
  29. 29.
    Uneyama H, Munakata M, Akaike N (1993) Caffeine response in pyramidal neurons freshly dissociated from rat hippocampus. Brain Res 604:24–31PubMedCrossRefGoogle Scholar
  30. 30.
    Ishibashi H, Akaike N (1995) Somatostatin modulates high-voltage-activated Ca2+ channels in freshly dissociated hippocampal neurons. J Neurophysiol 74:1028–1036PubMedGoogle Scholar
  31. 31.
    Ito Y, Murai Y, Ishibashi H, Onoue H, Akaike N (2000) The prostaglandin E series modulate high-voltage-activated calcium channels probably through the EP3 receptor in rat paratracheal ganglia. Neuropharmacology 39:181–190PubMedCrossRefGoogle Scholar
  32. 32.
    Barry PH, Lynch JW (1991) Liquid junction potentials and small cell effects in patch clamp analysis. J Membr Biol 121:101–117PubMedCrossRefGoogle Scholar
  33. 33.
    Kakazu Y, Uchida S, Nakagawa T, Akaike N, Nabekura J (2000) Reversibility and cation selectivity of the K+-Cl cotransport in rat central neurons. J Neurophysiol 84:281–288PubMedGoogle Scholar
  34. 34.
    Kakazu Y, Akaike N, Komiyama S, Nabekura J (1999) Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons. J Neurosci 19:2843–2851PubMedGoogle Scholar
  35. 35.
    Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A, Akaike N (2002) Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury. J Neurosci 22:4412–4417PubMedGoogle Scholar

Copyright information

© Springer 2012

Authors and Affiliations

  • Hitoshi Ishibashi
    • 1
  • Andrew J. Moorhouse
    • 2
  • Junichi Nabekura
    • 1
  1. 1.Department of Developmental PhysiologyNational Institute for Physiological Sciences, National Institutes of Natural SciencesOkazakiJapan
  2. 2.Department of Physiology, School of Medical SciencesUniversity of New South WalesSydneyAustralia

Personalised recommendations