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Channel opening locks agonist onto the GABAC receptor

Nature Neuroscience volume 2, pages 219–225 (1999)Cite this article

Abstract

Determination of the activation mechanism of neurotransmitter-operated ion channels has been hindered by a limited understanding of the relationship between agonist binding and the gating of the integral ion pore. Here we describe a [3H]ligand binding assay that enables us to make repeated binding measurements from the same intact oocyte expressing recombinant human ρ1 GABAC receptors and directly correlate the binding kinetics with electrophysiological measurements. We have determined an association rate for GABA of about 105 M–1s–1; this is four orders of magnitude slower than diffusion, indicating GABA has restricted access to its binding site. We also demonstrate that GABA dissociates at two rates. Our data are consistent with the faster rate being the true microscopic dissociation rate of GABA, with the slower rate occurring because the opening of the pore detains agonist release.

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Figure 1: Single oocyte binding method and comparison of the dose dependence of binding and activation.
Figure 2: Determination of the GABA dissociation rate.
Figure 3: GABA dissociation and receptor deactivation at low temperature.
Figure 4: Determination of the on rate for [3H]GABA.
Figure 5: Single-channel measurements.
Figure 6: The relationship between the maximum GABA-activated current and the deactivation time constant.

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References

  1. Auerbach, A. A statistical analysis of acetylcholine receptor activation in Xenopus myocytes: stepwise versus concerted models of gating. J. Physiol. (Lond.) 461, 339–378 ( 1993).

    Article  CAS  Google Scholar 

  2. Colquhoun, D. & Sakmann, B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. (Lond.) 369, 501– 557 (1985).

    Article  CAS  Google Scholar 

  3. Colquhoun, D. & Ogden, D. C. Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J. Physiol. (Lond.) 395, 131–159 ( 1988).

    Article  CAS  Google Scholar 

  4. Sine, S. M. & Steinbach, J. H. Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist. J. Physiol. (Lond.) 385, 325– 359 (1987).

    Article  CAS  Google Scholar 

  5. Weiss, D. S. & Magleby, K. L. Gating scheme for single GABA-activated Cl– channels determined from stability plots, dwell-time distributions, and adjacent-interval durations. J. Neurosci. 9, 1314–1324 (1989).

    Article  CAS  Google Scholar 

  6. Twyman, R. E., Rogers, C. J. & Macdonald, R. L. Intraburst kinetic properties of the GABAa receptor main conductance state of mouse spinal cord neurones in culture. J. Physiol. (Lond.) 423, 193– 220 (1990).

    Article  CAS  Google Scholar 

  7. Colquhoun, D. Imprecision in presentation of binding studies. Trends Pharmacol. Sci. 6, 197 (1985).

    Article  Google Scholar 

  8. Colquhoun, D. Binding, gating, affinity and efficacy. The interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br. J. Pharmacol. 125, 923–948 (1998).

    Article  Google Scholar 

  9. Colquhoun, D. & Sakmann, B. From muscle endplate to brain synapses: a short history of synapses and agonist-activated ion channels. Neuron 20, 381–387 ( 1998).

    Article  CAS  Google Scholar 

  10. Katz, B. & Miledi, R. The binding of acetylcholine to receptors and its removal from the synaptic cleft. J. Physiol. (Lond.) 231, 549–574 (1973).

    Article  CAS  Google Scholar 

  11. Colquhoun, D., Dionne, V. E., Steinbach, J. H. & Stevens, C. F. Conductance of channels opened by acetylcholine-like drugs in muscle endplate. Nature 253, 204–206 (1975).

    Article  CAS  Google Scholar 

  12. Sheridan, R. E. & Lester, H. A. Relaxation measurements on the acetylcholine receptor. Proc. Natl. Acad. Sci. USA 72, 3496–3500 (1975).

    Article  CAS  Google Scholar 

  13. Sheridan, R. E. & Lester, H. A. Functional stoichiometry at the nicotininc receptor: The photon cross section for phase 1 corresponds to two bis-q molecules per channel. J. Gen. Physiol. 80, 499–515 (1982).

    Article  CAS  Google Scholar 

  14. Amin, J. & Weiss, D. S. Homomeric ρ1 GABA channels: activation properties and domains. Receptors Channels 2, 227–236 (1994).

    CAS  PubMed  Google Scholar 

  15. Shimada, S., Cutting, G. & Uhl, G. R. γ-aminobutyric acid a or c receptor? γ-aminobutyric acid ρ1 receptor RNA induces bicuculline-, barbiturate-, and benzodiazepine-insensitive γ-aminobutyric acid responses in Xenopus oocytes. Mol. Pharmacol. 41, 683–687 (1992).

    CAS  PubMed  Google Scholar 

  16. Enna, S. J. & Snyder, S. H. Properties of γ-aminobutyric acid (GABA) binding in rat brain synaptic membrane fractions. Brain Res. 100, 81–97 ( 1975).

    Article  CAS  Google Scholar 

  17. Neubig, R. R. & Cohen, J. B. Permeability control by cholinergic receptors in Torpedo postsynaptic membranes: agonist dose-response relations measured at second and millisecond times. Biochemistry 19, 2770–2779 ( 1980).

    Article  CAS  Google Scholar 

  18. Olsen, R. W. Drug interactions at the GABA receptor-ionophore complex. Annu. Rev. Pharmacol. Toxicol. 22, 245–277 (1982).

    Article  CAS  Google Scholar 

  19. Chang, Y. & Weiss, D. S. Substitutions of the highly conserved M2 leucine create spontaneously opening ρ1 γ-aminobutyric acid receptors. Mol. Pharmacol. 53, 511– 523 (1998).

    Article  CAS  Google Scholar 

  20. Amin, J. & Weiss, D. S. Insights into the activation of ρ1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits. Proc. R. Soc. Lond. B 263, 273 –282 (1996).

    Article  CAS  Google Scholar 

  21. Maconochie, D., Zempel, J. & Steinbach, J. How quickly can GABAA receptors open? Neuron 12, 61–71 ( 1994).

    Article  CAS  Google Scholar 

  22. Lavoie, A. M., Tingey, J. J., Harrison, N. L., Pritchett, D. B. & Twyman, R. E. Activation and deactivation rates of recombinant GABAA receptor channels are dependent on α-subunit isoform. Biophys. J. 73, 2518–2526 (1997).

    Article  CAS  Google Scholar 

  23. Tia, S., Wang, J. F., Kotchabhakdi, N. & Vicini, S. Distinct deactivation and desensitization kinetics of recombinant GABA A receptors. Neuropharmacology 35, 1375–1382 (1996).

    Article  CAS  Google Scholar 

  24. Rang, H. P. The kinetics of action of acetylcholine antagonists in smooth muscle. Proc. R. Soc. Lond. B 164, 488–510 (1966).

    Article  CAS  Google Scholar 

  25. Thron, C. D. & Waud, D. R. The rate of action of atropine. J. Pharmacol. Exp. Ther. 160, 91– 105 (1968).

    CAS  PubMed  Google Scholar 

  26. Colquhoun, D., Large, W. A. & Rang, H. P. An analysis of the action of a false transmitter at the neuromuscular junction. J. Physiol. (Lond.) 266, 361–395 (1977).

    Article  CAS  Google Scholar 

  27. Armstrong, D. L. & Lester, H. A. The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft. J. Physiol. (Lond.) 294, 365– 386 (1979).

    Article  CAS  Google Scholar 

  28. Unwin, N. Acetylcholine receptor channel imaged in the open state. Nature 373, 37–43 ( 1995).

    Article  CAS  Google Scholar 

  29. Monod, J., Wyman, J. & Changeux, J. P. On the nature of allosteric proteins: a plausible model. J. Mol. Biol. 12, 88– 118 (1965).

    Article  CAS  Google Scholar 

  30. Haber, J. E. & Koshland, D. E. Relation of protein subunit interactions to the molecular species observed during cooperative binding of ligands. Proc. Natl. Acad. Sci. USA 58, 2087–2093 (1967).

    Article  CAS  Google Scholar 

  31. Liman, E. R., Tytgat, J. & Hess, P. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9, 861–871 ( 1992).

    Article  CAS  Google Scholar 

  32. Chang, Y., Wang, R., Barot, S. & Weiss, D. S. Stoichiometry of a recombinant GABAA receptor. J. Neurosci. 16, 5415–5424 (1996).

    Article  CAS  Google Scholar 

  33. Colquhoun, D. & Hawkes, A. G. On the stochastic properties of single ion channels. Proc. R. Soc. Lond. B 211, 205–235 (1981).

    Article  CAS  Google Scholar 

  34. Bean, B. P. ATP-activated channels in rat and bullfrog sensory neurons: concentration dependence and kinetics. J. Neurosci. 10, 1–10 (1990).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Robin A.J. Lester and Michael Quick for discussion. This work was supported by grants from the NIH (NS35291 and NS36196) and W.M. Keck Foundation (931360).

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  1. Departments of Neurobiology and Physiology and Biophysics, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue So., Birmingham, 35294-0021 , Alabama, USA

    Yongchang Chang & David S. Weiss

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Correspondence to David S. Weiss.

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Chang, Y., Weiss, D. Channel opening locks agonist onto the GABAC receptor. Nat Neurosci 2, 219–225 (1999). https://doi.org/10.1038/6313

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