Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

ABSTRACT Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA^sub A^ and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA^sub A^ receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.

INTRODUCTION

Conversion of an ion channel from a stable closed state to an open state is extremely rare unless an external force drives the channel open. At equilibrium, the ratio of closed to open channels defines the Gibbs free energy difference between the two states (Wentworth and Ladner, 1972). The external force shifts this ratio by altering the free energy difference. In voltage-gated channels, the electrostatic force associated with the transmembrane potential moves charges within the membrane, triggering the opening of the pore (Hille, 1992). The energy required for these movements can be calculated from the state transition rate constants measured in voltage jump experiments. Molecular and fluorescence techniques have been used to estimate the number and location of the charged residues and the distance that they move, providing a quantitative picture of voltage-dependent gating (Hille, 1992; Yang et al., 1996; Cha et al., 1999; Glauner et al., 1999).

The situation is much less clear for ligand-gated channels. From an experimental standpoint, it has been more difficult to make rapid agonist applications than rapid voltage steps. Thus, information about ligand gating has come largely from steady-state single channel records and from macroscopic dose-response curves using relatively slow ligand applications. Furthermore, gating charge movements, which are invaluable for studying gating steps in voltage-gated channels, especially those involving closed states, are not commonly observed in ligand-gated channels. Although molecular techniques have identified residues that may participate in binding and gating (Amin and Weiss, 1993; Schmieden et al., 1993; Xu and Akabas, 1996; Changeux and Edelstein, 1998; Paas, 1998; Wilson and Karlin, 1998; Boileau et al., 1999; Matulef et al., 1999; Wagner and Czajkowski, 2001) and electron diffraction measurements have revealed the structure of a ligand-gated channel to 4.6-A resolution (Miyazawa et al., 1999), such methods provide a relatively static picture of channel structure. In contrast, these channels normally function under highly nonequilibrium conditions. Kinetic studies thus provide a valuable link in understanding the relationship between ligand binding and channel gating.

A few common themes have emerged from studies of several families of ligand-gated channels. For example, agonist binding appears to involve multiple, discontinuous protein domains, often from separate receptor subunits (Dennis et al., 1988; Schmieden et al., 1992; Vandenberg et al., 1992; Amin and Weiss, 1993; Stem-Bach et al., 1994; Paas, 1998; Boileau et al., 1999; Wagner and Czajkowski, 2001). Binding could thus involve a type of chelation or "induced fit" process (Koshland et al., 1966; Fersht, 1985) in which separate regions of the receptor come together to interact with the agonist. A chelation mechanism implies that the agonist may reciprocally organize separate regions of the receptor into a relatively rare conformation such as an open state. Such reciprocal interactions between agonist and receptor are likely because channel opening is rare in the absence of agonist (Jackson, 1984), but when channels are open, agonists can be trapped at the binding site (Benveniste and Mayer, 1995; Chang and Weiss, 1999).

Agonist binding rates are often slower than expected for a diffusion-limited process (Sine and Steinbach, 1986; Zhang et al., 1995; Akk and Auerbach, 1996; Jones et al., 1998), implying that an energy-requiring process precedes or accompanies binding (Jones et al., 1998). Under the agonist chelation hypothesis, this process would correspond to structural rearrangements in the binding site that lead to channel opening. This hypothesis therefore predicts that ligands capable of opening the channel must bind slower than the diffusion limit, whereas ligands that do not open the channel (i.e., competitive antagonists) should bind more rapidly than agonists.

MATERIALS AND METHODS

Slice preparation and electrophysiology

We thank Drs. Sanjive Qazi and Jan Behrends for helpful comments on the manuscript, and Dr. Boris Barbour and three anonymous reviewers for helpful criticism.

M.V.J. was sponsored in part by the American Epilepsy Society with support from the Milken Family Medical Foundation. Y.S. was supported by a Core Research for Evolutional Science and Technology program from the Japanese Science and Technology Corporation. This work was supported by National Institutes of Health grant NS26494 (G.L.W.), Deutsche Forschungsgemeinschaft grant Jo-248/2-1 (P.J.), and a grant from the Human Frontiers Research Program Organization.

[Reference]

REFERENCES

[Reference]

Akk, G., and A. Auerbach. 1996. Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors. Biophys. J. 70:2652-2658.

Amin, J., and D. S. Weiss. 1993. GABAA receptor needs two homologous domains of the beta-subunit for activation by GABA but not by pentobarbital. Nature. 366:565-569.

Andrews, P. R., and G. A. R. Johnston. 1979. GABA agonists and antagonists. Biochem. Pharmacol. 28:2697-2702.

Balmer, L. 1997. Signals and Systems: An Introduction. (2nd ed.) PrenticeHall, Upper Saddle River, NJ. 118-171.

Benveniste, M. and M. L. Mayer. 1995. Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine. J. PhysioL (Lond.). 483:367-384.

Boileau, A. J., A. R. Evers, A. F. Davis, and C. Czajkowski. 1999. Mapping the agonist binding site of the GABAA receptor: evidence for a 0-strand. J. Neurosci. 19:4874-4854.

[Reference]

Bormann, J., and D. E. Clapham. 1985. -y-Aminobutyric acid receptor channels in adrenal chromaffin cells: a patch-clamp study. Proc. Natl. Acad. Sci. U.S.A. 82:2168-2172.

Cha, A., G. E. Snyder, P. R. Selvin, and F. Bezanilla. 1999. Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature. 402:809-813.

Chambon, J. P., P. Feltz, M. Heaulme, S. Restle, R. Schlichter, K. Biziere, and C. G. Wermuth. 1985. An arylaminopyridazine derivative of y-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor. Proc. Natl. Acad. Sci. U.S.A. 82:1832-1836.

Chang, Y., and D. Weiss. 1999. Channel opening locks agonist onto the GABAc receptor. Nat. Neurosci. 2:219-225.

Changeux, J.-P., and S. J. Edelstein. 1998. Allosteric receptors after 30 years. Neuron. 21:959-980.

Constanti, A. 1977. Comparison of dose/conductance curves for GABA and some structurally related compounds at the lobster inhibitory neuromuscular junction. Neuropharmacology. 16:367-374.

[Reference]

Dennis, M., J. Giraudat, F. Kotzyba-Hibert, M. Goeldner, C. Hirth, J. Y. Chang, C. Lazure, M. Chretien, and J. P. Changeux. 1988. Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry. 27:2346-2357.

Duittoz, A. H., and R. J. Martin. 1991. Antagonist properties of arylaminopyridazine GABA derivatives at the Ascaris muscle GABA receptor. J. Exp. Biol. 159:149-164.

Fersht, A. 1985. Enzyme Structure and Mechanism. (2nd ed.) W.H. Freeman and Co., New York. 263-346.

Glauner, K. S., L. M. Mannuzzu, C. S. Handhi, and E. Y. Isacoff. 1999. Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature. 402:813-817.

Hamann, H., M. Desarmenien, E. Desaulles, M. F. Bader, and P. Feltz. 1988. Quantitative evaluation of the properties of a pyridazinyl GABA

[Reference]

derivative (SR 95531) as a GABAA competitive antagonist. An electrophysiological approach. Brain Res. 442:287-296.

Hille, B. 1992. Ionic Channels of Excitable Membranes (2nd ed.) Sinauer Associates, Inc., Sunderland, MA. 423-503.

Jackson, M. B. 1984. Spontaneous openings of the acetylcholine receptor channel. Proc. Natl. Acad. Sci. U.S.A. 81:3901-3904.

Jonas, P., J. Bischofberger, and J. SandkUhler. 1998. Corelease of two fast neurotransmitters at a central synapse. Science. 281:419-424.

Jones, M. V., Y. Sahara, J. A. Dzubay, and G. L. Westbrook. 1998. Defining affinity with the GABAA receptor. J. Neurosci. 18: 8590-8604.

Jones, M. V., and G. L. Westbrook. 1997. Shaping of IPSCs by endogenous calcineurin activity. J. Neurosci. 17:7626-7633.

[Reference]

Kemp, J. A., G. R. Marshall, and G. N. Woodruff. 1986. Quantitative evaluation of the potencies of GABA-receptor agonists and antagonists using the rat hippocampal slice preparation. Br. J. PharmacoL 87: 677-684.

Koshland, D. E., Jr., G. Nemethy, and D. Filmer. 1966. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 5:365-385.

Lauffer, M. A. 1989. Motion in Biological Systems. Alan R. Liss, Inc., New York. 23-58.

Levy, S. 1995. Three dimensional geometry: quadrics. In Standard Mathematical Tables and Formulae. (30th ed.). D. Zwillinger, editor. CRC Press, Boca Raton, FL. http://www.geom.umn.edu/docs/reference/CRCformulas/book.html.

Lew, M. J., and J. A. Angus. 1995. Analysis of competitive agonist-antagonist interactions by nonlinear regression. Trends. Pharm. Sci. 16:328-337.

Macdonald, R. L., C. J. Rogers, and R. E. Twyman. 1989. Kinetic properties of the GABAA receptor main conductance state of mouse spinal neurones in culture. J. Physiol. (Land.). 410:479-499.

[Reference]

Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels. Neuron. 24:443-452.

Miyazawa, A., Y. Fujiyoshi, M. Stowell, and N. Unwin. 1999. Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall. J. MoL Biol. 288:765-786.

Motulsky, H. J., and L. A. Ransnas. 1987. Fitting curves to data using nonlinear regression: a practical and nonmathematical review. FASEB J. 1:365-374.

Olsen, R. W. 1982. Drug interactions at the GABA receptor-ionophore complex. Ann. Rev. Pharmacol. ToxicoL 22:245-277.

Olsen, R. W., D. M. Sapp, M. H. Bureau, D. M. Turner, and N. Kokka. 1991. Allosteric actions of central nervous system depressants including

[Reference]

anesthetics on subtypes of the inhibitory y-aminobutyric acid" receptorchloride channel complex. Ann. MY Acad. Sci. 625:145-154.

Paas, Y. 1998. The macro- and microarchitectures of the ligand-binding domain of glutamate receptors. Trends Neurosci. 21:117-125. Ragozzino, D., R. M. Woodward, Y. Murata, F. Eusebi, L. E. Overman,

and R. Miledi. 1996. Design and in vitro pharmacology of a selective y-aminobutyric acid, receptor antagonist. Mol. Pharmacol. 50: 1024-1030.

Schmieden, V., J. Kuhse, and H. Betz. 1992. Agonist pharmacology of neonatal and adult glycine receptor a subunits: identification of amino acid residues involved in taurine activation. EMBO J. 11:2025-2032.

Schmieden, V., J. Kuhse, and H. Betz. 1993. Mutation of glycine receptor subunit creates 0-alanine receptor responsive to GABA. Science. 262: 256-258.

[Reference]

Sine, S. M., and J. H. Steinbach. 1986. Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist. J. Physiol (Lord.). 373:129-162.

Stem-Bach, Y., B. Bettler, M. Hartley, P. O. Sheppard, P. J. O'Hara, and S. F. Heinemann. 1994. Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acidbinding proteins. Neuron. 13:1345-1357.

Twyman, R. E., C. J. Rogers, and R. L. Macdonald. 1990. Intraburst kinetic properties of the GABAA receptor main conductance state of mouse spinal chord neurones in culture. J. Physiol. (Lond.). 423:193-220.

Ueno, S., J. Bracamontes, C. Zorumski, D. S. Weiss, and J. H. Steinbach. 1997. Bicuculline and gabazine are allosteric inhibitors of channel opening of the GABAA receptor. J. Neurosci. 17:625-634.

Vandenberg, R. J., C. A. Handford, and P. R. Schofield. 1992. Distinct agonist- and antagonist-binding sites on the glycine receptor. Neuron. 9:491-496.

Wagner, D. A., and C. Czajkowski. 2001. Structure and dynamics of the GABA binding pocket: a narrowing cleft that constricts during activation. J. Neurosci. 21:67-74.

[Reference]

Wentworth, W. E., and S. J. Ladner. 1972. Fundamentals of Physical Chemistry. Wadsworth Publishing Co., Belmont, CA. 223-280.

Wilson, G. G. and Karlin, A. 1998. The location of the gate in the acetylcholine receptor channel. Neuron. 20:1269-1281.

Xu, M., and M. H. Akabas. 1996. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAA receptor al subunit. J. Gen. Physiol. 107:195-205.

Yang, N., A. L. J. George, and R. Horn. 1996. Molecular basis of charge movement in voltage-gated sodium channels. Neuron. 16:113-112. Zhang, Y., J. Chen, and A. Auerbach. 1995. Activation of recombinant

mouse acetylcholine receptors by acetylcholine, carbamylcholine and tetramethylammonium. J. PhysioL (Loud.). 486:189-206.

[Author Affiliation]

Mathew V. Jones,* Peter Jonas,^ Yoshinori Sahara,^^ and Gary L. Westbrook(sec)

[Author Affiliation]

*Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706 USA, ^Physiologisches Institut der Universitat Freiburg, D-79104 Freiburg, Germany, ^^Department of Maxillofacial Biology, Tokyo Medical and Dental University, 113 Tokyo, Japan, and (sec)Vollum Institute and Department of Neurology, Oregon Health Sciences University, Portland, Oregon 97201 USA

[Author Affiliation]

Received for publication 28 March 2001 and in final form 27 July 2001. Address reprint requests to Mathew V. Jones, Dept. of Physiology, Univ. of Wisconsin-Madison, 127 SMI, 1300 University Ave., Madison, WI 53706-1510. Tel.: 608-263-4495; Fax: 608-263-6120; E-mail: jonesmat@physiology.wisc.edu.