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Recording and Hodgkin-Huxley Kinetic Analysis of Voltage-Gated Ion Channels in Nucleated Patches

  • Mara Almog
  • Alon KorngreenEmail author
Protocol
Part of the Neuromethods book series (NM, volume 113)

Abstract

The patch-clamp technique is widely used to measure and characterize ion currents flowing through the cell membrane. Various configurations can be used according to the research hypothesis. A relatively new configuration of the patch-clamp technique is the outside-out nucleated patch, which allows measuring somatic currents in voltage-clamp recordings due to a reduction in technical issues. Characterization of ionic currents is mostly based on the Hodgkin-Huxley model, a detailed mathematical and biophysical model of membrane excitability. This chapter describes voltage-clamp experiments step by step, from preparation and performing outside-out nucleated patch experiments through to the Hodgkin-Huxley analysis of the recorded data.

Key words

Nucleated patch Outside-out patch Voltage-gated channels Patch clamp Kinetic analysis Hodgkin-Huxley model 

Notes

Acknowledgments

This work was supported by a grant from the German-Israeli Foundation to AK (#1091-27.1/2010).

References

  1. 1.
    Neher E, Sakmann B, Steinbach JH (1978) The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch 375(2):219–228CrossRefPubMedGoogle Scholar
  2. 2.
    Edwards FA, Konnerth A, Sakmann B, Takahashi T (1989) A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch 414(5):600–612CrossRefPubMedGoogle Scholar
  3. 3.
    Stuart GJ, Dodt HU, Sakmann B (1993) Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflugers Arch 423(5-6):511–518CrossRefPubMedGoogle Scholar
  4. 4.
    Sigworth F (1995) Design of the EPC-9, a computer-controlled patch-clamp amplifier. 1. Hardware. J Neurosci Methods 56(2):195–202CrossRefGoogle Scholar
  5. 5.
    Johnston D, Wu SM-s (1995) Foundations of cellular neurophysiology. MIT Press, Cambridge, MAGoogle Scholar
  6. 6.
    Sigworth FJ, Affolter H, Neher E (1995) Design of the EPC-9, a computer-controlled patch-clamp amplifier. 2. Software. J Neurosci Methods 56(2):203–215CrossRefPubMedGoogle Scholar
  7. 7.
    Hodgkin AL, Huxley AF (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117(4):500–544CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hodgkin AL, Huxley AF, Katz B (1952) Measurement of current-voltage relations in the membrane of the giant axon of Loligo. J Physiol 116(4):424–448CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Schaefer AT, Helmstaedter M, Schmitt AC, Bar-Yehuda D, Almog M, Ben-Porat H, Sakmann B, Korngreen A (2007) Dendritic voltage-gated K+ conductance gradient in pyramidal neurones of neocortical layer 5B from rats. J Physiol 579(Pt 3):737–752CrossRefPubMedGoogle Scholar
  10. 10.
    Schaefer AT, Helmstaedter M, Sakmann B, Korngreen A (2003) Correction of conductance measurements in non-space-clamped structures: 1. Voltage-gated K+ channels. Biophys J 84(6):3508–3528CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bar-Yehuda D, Korngreen A (2008) Space-clamp problems when voltage clamping neurons expressing voltage-gated conductances. J Neurophysiol 99(3):1127–1136CrossRefPubMedGoogle Scholar
  12. 12.
    Chen X, Whissell P, Orser BA, MacDonald JF (2011) Functional modifications of acid-sensing ion channels by ligand-gated chloride channels. PLoS One 6(7):e21970CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kim NK, Robinson HP (2011) Effects of divalent cations on slow unblock of native NMDA receptors in mouse neocortical pyramidal neurons. Eur J Neurosci 34(2):199–212CrossRefPubMedGoogle Scholar
  14. 14.
    Sather W, Dieudonne S, MacDonald JF, Ascher P (1992) Activation and desensitization of N-methyl-D-aspartate receptors in nucleated outside-out patches from mouse neurones. J Physiol 450:643–672CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Almog M, Korngreen A (2009) Characterization of voltage-gated Ca2+ conductances in layer 5 neocortical pyramidal neurons from rats. PLoS One 4(4), e4841CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bekkers JM (2000) Properties of voltage-gated potassium currents in nucleated patches from large layer 5 cortical pyramidal neurons of the rat. J Physiol 525(Pt 3):593–609CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jung SC, Eun SY (2012) Sustained K+ outward currents are sensitive to intracellular heteropodatoxin2 in CA1 neurons of organotypic cultured hippocampi of rats. Kr J Physiol Pharmacol 16(5):343–348CrossRefGoogle Scholar
  18. 18.
    Khurana S, Liu Z, Lewis AS, Rosa K, Chetkovich D, Golding NL (2012) An essential role for modulation of hyperpolarization-activated current in the development of binaural temporal precision. J Neurosci 32(8):2814–2823CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Korngreen A, Sakmann B (2000) Voltage-gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients. J Physiol 525(Pt 3):621–639CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lin YC, Liu YC, Huang YY, Lien CC (2010) High-density expression of Ca2+-permeable ASIC1a channels in NG2 glia of rat hippocampus. PLoS One 5(9)Google Scholar
  21. 21.
    Gentet LJ, Stuart GJ, Clements JD (2000) Direct measurement of specific membrane capacitance in neurons. Biophys J 79(1):314–320CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Veruki ML, Oltedal L, Hartveit E (2010) Electrical coupling and passive membrane properties of AII amacrine cells. J Neurophysiol 103(3):1456–1466CrossRefPubMedGoogle Scholar
  23. 23.
    Neher E (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207:123–131CrossRefPubMedGoogle Scholar
  24. 24.
    Barry PH, Diamond JM (1970) Junction potentials, electrode standard potentials, and other problems in interpreting electrical properties of membranes. J Membr Biol 3(1):93–122CrossRefPubMedGoogle Scholar
  25. 25.
    Barry PH, Lynch JW (1991) Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 121(2):101–117CrossRefPubMedGoogle Scholar
  26. 26.
    Costantin JL, Qin N, Waxham MN, Birnbaumer L, Stefani E (1999) Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A. Pflugers Arch 437(6):888–894CrossRefPubMedGoogle Scholar
  27. 27.
    Josephson IR, Varadi G (1996) The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels. Biophys J 70(3):1285–1293CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Belles B, Hescheler J, Trautwein W, Blomgren K, Karlsson JO (1988) A possible physiological role of the Ca2+-dependent protease calpain and its inhibitor calpastatin on the Ca2+ current in guinea pig myocytes. Pflugers Arch 412(5):554–556CrossRefPubMedGoogle Scholar
  29. 29.
    Romanin C, Grosswagen P, Schindler H (1991) Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches. Pflugers Arch 418(1-2):86–92CrossRefPubMedGoogle Scholar
  30. 30.
    Ikeda SR (1991) Double-pulse calcium channel current facilitation in adult rat sympathetic neurones. J Physiol 439:181–214CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Scamps F, Valentin S, Dayanithi G, Valmier J (1998) Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons. Neuroscience 87(3):719–730CrossRefPubMedGoogle Scholar
  32. 32.
    Hodgkin AL, Huxley AF (1952) The components of membrane conductance in the giant axon of Loligo. J Physiol 116(4):473–496CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Hodgkin AL, Huxley AF (1952) The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J Physiol 116(4):497–506CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Connor JA, Stevens CF (1971) Voltage clamp studies of a transient outward membrane current in gastropod neural somata. J Physiol 213(1):21–30CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Gurkiewicz M, Korngreen A (2006) Recording, analysis, and function of dendritic voltage-gated channels. Pflugers Arch 453(3):283–292CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.The Mina and Everard Goodman Faculty of Life SciencesBar-Ilan UniversityRamat-GanIsrael
  2. 2.Leslie and Susan Gonda Multidisciplinary Brain Research CenterBar-Ilan UniversityRamat-GanIsrael

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