Skip to main content
SpringerLink
Log in

Amplitude Histogram-Based Method of Analysis of Patch Clamp Recordings that Involve Extreme Changes in Channel Activity Levels

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Many ion channels show low basal activity, which is increased hundreds-fold by the relevant gating factor. A classical example is the activation G-protein-activated K+ channels (GIRK) by Gβγ subunit dimer. The extent of activation (relative to basal current), R a, is an important physiological parameter, usually readily estimated from whole cell recordings. However, calculation of R a often becomes non-trivial in multi-channel patches because of extreme changes in activity upon activation, from a seemingly single-channel pattern to a macroscopic one. In such cases, calculation of the net current flowing through the channels in the patch, \(\overline I \), before and after activation may require different methods of analysis. To address this problem, we utilized neuronal GIRK channels activated by purified Gβγ in excised patches of Xenopus oocytes. Channels were expressed at varying densities, from a few to several hundreds per patch. We present a simple and fast method of calculating \(\overline I \) using amplitude histogram analysis and establish its accuracy by comparing with \(\overline I \) calculated from event lists. This method allows the analysis of extreme changes in \(\overline I \) in multichannel patches, which would be impossible using the standard methods of idealization and event list generation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  • Alvarez, O., Gonzalez, C., & Latorre, R. (2002). Counting channels: A tutorial guide on ion channel fluctuation analysis. Advances in Physiology Education, 26, 327–341.

    PubMed  Google Scholar 

  • Bauer, R. J., Bowman, B. F., & Kenyon, J. L. (1987). Theory of the kinetic analysis of patch-clamp data. Biophysical Journal, 52, 961–978.

    Article  PubMed  CAS  Google Scholar 

  • Blunck, R., Kirst, U., Riessner, T., & Hansen, U. (1998). How powerful is the dwell-time analysis of multichannel records. The Journal of Membrane Biology, 165, 19–35. doi:10.1007/s002329900417.

    Article  PubMed  CAS  Google Scholar 

  • Colquhoun, D., & Hawkes, A. G. (1977). Relaxation and fluctuations of membrane currents that flow through drug-operated channels. Proceedings of the Royal Society of London. Series B Biological Sciences, 199, 231–262.

    CAS  Google Scholar 

  • Colquhoun, D., & Hawkes, A. G. (1981). On the stochastic properties of single ion channels. Proceedings of the Royal Society of London. Series B Biological Sciences, 211, 205–235.

    CAS  Google Scholar 

  • Colquhoun, D., & Hawkes, A. G. (1995). The principles of the stochastic interpretation of ion-channel mechanisms. In B. Sakmann, & E. Neher (Eds.), Single-channel recording (pp. 397–482). New York: Plenum.

    Google Scholar 

  • Dascal, N. (1997). Signalling via the G protein-activated K+ channels. Cellular Signalling, 9, 551–573. doi:10.1016/S0898-6568(97)00095-8.

    Article  PubMed  CAS  Google Scholar 

  • Grigg, J. J., Kozasa, T., Nakajima, Y., & Nakajima, S. (1996). Single-channel properties of a G-protein-coupled inward rectifier potassium channel in brain neurons. Journal of Neurophysiology, 75, 318–328.

    PubMed  CAS  Google Scholar 

  • Ho, I. H., & Murrell-Lagnado, R. D. (1999). Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels. The Journal of Physiology, 520(Pt 3), 645–651. doi:10.1111/j.1469-7793.1999.00645.x.

    Article  PubMed  CAS  Google Scholar 

  • Hosoya, Y., Yamada, M., Ito, H., & Kurachi, Y. (1996). A functional model for G protein activation of the muscarinic K+ channel in guinea pig atrial myocytes. Spectral analysis of the effect of GTP on single-channel kinetics. The Journal of General Physiology, 108, 485–495. doi:10.1085/jgp.108.6.485.

    Article  PubMed  CAS  Google Scholar 

  • Howe, J. R., Cull-Candy, S. G., & Colquhoun, D. (1991). Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells. The Journal of Physiology, 432, 143–202.

    PubMed  CAS  Google Scholar 

  • Ivanina, T., Varon, D., Peleg, S., Rishal, I., Porozov, Y., Dessauer, C. W., et al. (2004). Gαi1 and Gαi3 differentially interact with, and regulate, the G protein-activated K+ channel. The Journal of Biological Chemistry, 279, 17260–17268. doi:10.1074/jbc.M313425200.

    Article  PubMed  CAS  Google Scholar 

  • Jackson, M. B. (1992). Ion channels. Single-channel analysis. Methods in Enzymology, 207, 729–746. doi:10.1016/0076-6879(92)07053-Q.

    Article  PubMed  CAS  Google Scholar 

  • Jelacic, T. M., Sims, S. M., & Clapham, D. E. (1999). Functional expression and characterization of G-protein-gated inwardly rectifying K+ channels containing GIRK3. The Journal of Membrane Biology, 169, 123–129. doi:10.1007/s002329900524.

    Article  PubMed  CAS  Google Scholar 

  • Neher, E., & Stevens, C. F. (1977). Conductance fluctuations and ionic pores in membranes. Annual Review of Biophysics and Bioengineering, 6, 345–381. doi:10.1146/annurev.bb.06.060177.002021.

    Article  PubMed  CAS  Google Scholar 

  • Nemec, J., Wickman, K., & Clapham, D. E. (1999). Gβγ binding increases the open time of IKACh: Kinetic evidence for multiple Gβγ binding sites. Biophysical Journal, 76, 246–252.

    Article  PubMed  CAS  Google Scholar 

  • Peleg, S., Varon, D., Ivanina, T., Dessauer, C. W., & Dascal, N. (2002). Gαi controls the gating of the G protein-activated K+ channel, GIRK. Neuron, 33, 87–99. doi:10.1016/S0896-6273(01)00567-0.

    Article  PubMed  CAS  Google Scholar 

  • Qin, F., Auerbach, A., & Sachs, F. (1996). Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophysical Journal, 70, 264–280.

    Article  PubMed  CAS  Google Scholar 

  • Qin, F., Auerbach, A., & Sachs, F. (1997). Maximum likelihood estimation of aggregated Markov processes. Proc Biol Sci, 264, 375–383. doi:10.1098/rspb.1997.0054.

    Article  PubMed  CAS  Google Scholar 

  • Qin, F., Auerbach, A., & Sachs, F. (2000a). A direct optimization approach to hidden Markov modeling for single channel kinetics. Biophysical Journal, 79, 1915–1927.

    Article  PubMed  CAS  Google Scholar 

  • Qin, F., Auerbach, A., & Sachs, F. (2000b). Hidden Markov modeling for single channel kinetics with filtering and correlated noise. Biophysical Journal, 79, 1928–1944.

    Article  PubMed  CAS  Google Scholar 

  • Rishal, I., Keren-Raifman, T., Yakubovich, D., Ivanina, T., Dessauer, C. W., Slepak, V. Z., et al. (2003). Na+ promotes the dissociation between Gα-GDP and Gβγ, activating G protein-gated K+ channels. The Journal of Biological Chemistry, 278, 3840–3845. doi:10.1074/jbc.C200605200.

    Article  PubMed  CAS  Google Scholar 

  • Sachs, F., Neil, J., & Barkakati, N. (1982). The automated analysis of data from single ionic channels. Pflugers Archiv, 395, 331–340. doi:10.1007/BF00580798.

    Article  PubMed  CAS  Google Scholar 

  • Schreibmayer, W., Dessauer, C. W., Vorobiov, D., Gilman, A. G., Lester, H. A., Davidson, N., et al. (1996). Inhibition of an inwardly rectifying K+ channel by G-protein α-subunits. Nature, 380, 624–627. doi:10.1038/380624a0.

    Article  PubMed  CAS  Google Scholar 

  • Traynelis, S. F., & Jaramillo, F. (1998). Getting the most out of noise in the central nervous system. Trends in Neurosciences, 21, 137–145. doi:10.1016/S0166-2236(98)01238-7.

    Article  PubMed  CAS  Google Scholar 

  • Vivaudou, M. B., Singer, J. J., & Walsh Jr., J. V. (1986). An automated technique for analysis of current transitions in multilevel single-channel recordings. Pflugers Archiv, 407, 355–364. doi:10.1007/BF00652618.

    Article  PubMed  CAS  Google Scholar 

  • Yakubovich, D., Pastushenko, V., Bitler, A., Dessauer, C. W., & Dascal, N. (2000). Slow modal gating of single G protein-activated K+ channels expressed in Xenopus oocytes. The Journal of Physiology, 524(Pt 3), 737–755. doi:10.1111/j.1469-7793.2000.00737.x.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from NIH [GM68493 (N.D.) and GM60419 (C.W.D.)] and US–Israel Binational Science Foundation (01-122, N.D. and C.W.D). The authors report no conflicts of interest.

Author information

Author notes

  1. Ida Rishal

    Present address: Weizmann Institute of Science, Rehovot, Israel

Authors and Affiliations

  1. Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel

    Daniel Yakubovich, Ida Rishal, Carmen W. Dessauer & Nathan Dascal

  2. Department of Integrative Biology and Pharmacology, Medical School, University of Texas, Houston, TX, 77030, USA

    Carmen W. Dessauer

Authors
  1. Daniel Yakubovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ida Rishal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carmen W. Dessauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nathan Dascal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nathan Dascal.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 25.0 KB)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yakubovich, D., Rishal, I., Dessauer, C.W. et al. Amplitude Histogram-Based Method of Analysis of Patch Clamp Recordings that Involve Extreme Changes in Channel Activity Levels. J Mol Neurosci 37, 201–211 (2009). https://doi.org/10.1007/s12031-008-9117-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12031-008-9117-z

Keywords

Search

Navigation