Article | Published:

Zinc-mediated inhibition of GABAA receptors: discrete binding sites underlie subtype specificity

Abstract

Zinc ions are concentrated in the central nervous system and regulate GABAA receptors, which are pivotal mediators of inhibitory synaptic neurotransmission. Zinc ions inhibit GABAA receptor function by an allosteric mechanism that is critically dependent on the receptor subunit composition: αβ subunit combinations show the highest sensitivity, and αβγ isoforms are the least sensitive. Here we propose a mechanistic and structural basis for this inhibition and its dependence on the receptor subunit composition. We used molecular modeling to identify three discrete sites that mediate Zn2+ inhibition. One is located within the ion channel, and the other two are on the external amino (N)-terminal face of the receptor at the interfaces between α and β subunits. We found that the characteristically low Zn2+ sensitivity of GABAA receptors containing the γ2 subunit results from disruption to two of the three sites after receptor subunit co-assembly.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Frederickson, C.J. Neurobiology of zinc and zinc-containing neurons. Int. Rev. Neurobiol. 31, 145–238 (1989).

  2. 2

    Smart, T.G., Xie, X. & Krishek, B.J. Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Prog. Neurobiol. 42, 393–341 (1994).

  3. 3

    Frederickson, C.J. & Bush, A.I. Synaptically released zinc: physiological functions and pathological effects. Biometals 14, 353–366 (2001).

  4. 4

    Draguhn, A., Verdoorn, T.A., Ewert, M., Seeburg, P.H. & Sakmann, B. Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2+. Neuron 5, 781–788 (1990).

  5. 5

    Smart, T.G., Moss, S.J., Xie, X. & Huganir, R.L. GABAA receptors are differentially sensitive to zinc: dependence on subunit composition. Br. J. Pharmacol. 103, 1837–1839 (1991).

  6. 6

    Smart, T.G. A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones. J. Physiol (Lond.) 447, 587–625 (1992).

  7. 7

    Taketo, M. & Yoshioka, T. Developmental change of GABAA receptor-mediated current in rat hippocampus. Neuroscience 96, 507–514 (2000).

  8. 8

    Brooks-Kayal, A.R. et al. γ-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development. J. Neurochem. 77, 1266–1278 (2001).

  9. 9

    Buhl, E.H., Otis, T.S. & Mody, I. Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model. Science 271, 369–373 (1996).

  10. 10

    Gibbs, J.W. III, Shumate, M.D. & Coulter, D.A. Differential epilepsy-associated alterations in postsynaptic GABAA receptor function in dentate granule and CA1 neurons. J. Neurophysiol. 77, 1924–1938 (1997).

  11. 11

    Molnar, P. & Nadler, J.V. Lack of effect of mossy fiber-released zinc on granule cell GABAA receptors in the pilocarpine model of epilepsy. J. Neurophysiol. 85, 1932–1940 (2001).

  12. 12

    Rabow, L.E., Russek, S.J. & Farb, D.H. From ion currents to genomic analysis: recent advances in GABAA receptor research. Synapse 21, 189–274 (1996).

  13. 13

    Sieghart, W. Structure and pharmacology of γ-Aminobutyric acidA receptor subtypes. Pharmacol. Rev. 47, 181–234 (1995).

  14. 14

    Mehta, A.K. & Ticku, M.K. An update on GABAA receptors. Brain Res. Rev. 29, 196–217 (1999).

  15. 15

    Moss, S.J. & Smart, T.G. Constructing inhibitory synapses. Nat. Rev. Neurosci. 2, 240–250 (2001).

  16. 16

    Saxena, N.C. & MacDonald, R.L. Properties of putative cerebellar y-Aminobutyric acid A receptor isoforms. Mol. Pharmacol. 49, 567–579 (1996).

  17. 17

    Krishek, B.J., Moss, S.J. & Smart, T.G. Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors. J. Physiol. 507, 639–652 (1998).

  18. 18

    Whiting, P.J. et al. Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties. J. Neurosci. 17, 5027–6037 (1997).

  19. 19

    Celentano, J.J., Gyenes, M., Gibbs, T.T. & Farb, D.H. Negative modulation of the γ-aminobutyric acid response by extracellular zinc. Mol. Pharmacol. 40, 766–773 (1991).

  20. 20

    Legendre, P. & Westbrook, G.L. Noncompetitive inhibition of γ-Aminobutyric acidA channels by Zn. Mol. Pharmacol. 39, 267–274 (1991).

  21. 21

    Gingrich, K.J. & Burkat, P.M. Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the gamma-subunit. J. Physiol. 506, 609–625 (1998).

  22. 22

    Vallee, B.L. & Auld, D.S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647–5659 (1990).

  23. 23

    Auld, D.S. Zinc coordination sphere in biochemical zinc sites. Biometals 14, 271–313 (2001).

  24. 24

    Wooltorton, J.R., McDonald, B.J., Moss, S.J. & Smart, T.G. Identification of a Zn2+ binding site on the murine GABAA receptor complex: dependence on the second transmembrane domain of β subunits. J. Physiol. 505, 633–640 (1997).

  25. 25

    Horenstein, J. & Akabas, M.H. Location of a high affinity Zn2+ binding site in the channel of α1β1 γ-aminobutyric acidA receptors. Mol. Pharmacol. 53, 870–877 (1998).

  26. 26

    Dunne, E.L. et al. An N-terminal histidine regulates Zn2+ inhibition on the murine GABAA receptor β3 subunit. Br. J. Pharmacol. 137, 29–38 (2002).

  27. 27

    Barnard, E.A., Darlison, M.G. & Seeburg, P. Molecular biology of GABAA receptor: the receptor/channel superfamily. Trends Neurosci. 10, 502–509 (1987).

  28. 28

    Wang, T.-L., Hackam, A., Guggino, W.B. & Cutting, G.R. A single histidine residue is essential for zinc inhibition of GABA p1 receptors. J. Neurosci. 15, 7684–7691 (1996).

  29. 29

    Wooltorton, J.R., Moss, S.J. & Smart, T.G. Pharmacological and physiological characterization of murine homomeric β3 GABAA receptors. Eur. J. Neurosci. 9, 2225–2235 (1997).

  30. 30

    Brejc, K. et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276 (2001).

  31. 31

    Horenstein, J., Wagner, D.A., Czajkowski, C. & Akabas, M.H. Protein mobility and GABA-induced conformational changes in GABAA receptor pore-lining M2 segment. Nat. Neurosci. 4, 477–485 (2001).

  32. 32

    Nagaya, N. & MacDonald, R.L. Two γ2L subunit domains confer low Zn2+ sensitivity to ternary GABAA receptors. J. Physiol. 532, 17–30 (2001).

  33. 33

    Tretter, V., Ehya, N., Fuchs, K. & Sieghart, W. Stoichiometry and assembly of a recombinant GABAA receptor subtype. J. Neurosci. 17, 2728–2737 (1997).

  34. 34

    Farrar, S.J., Whiting, P.J., Bonnert, T.P. & McKernan, R.M. Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. J. Biol. Chem. 274, 10100–10104 (1999).

  35. 35

    Newell, J.G. & Dunn, S.M. Functional consequences of the loss of high affinity agonist binding to gamma-aminobutyric acid type-A receptors. Implications for receptor desensitization. J. Biol. Chem. 277, 21423–21430 (2002).

  36. 36

    Amin, J. & Weiss, D.S. GABAA receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital. Nature 366, 565–569 (1993).

  37. 37

    Smith, G.B. & Olsen, R.W. Functional domains of GABAA receptors. Trends Pharmacol. Sci. 16, 162–168 (1995).

  38. 38

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

  39. 39

    Boileau, A.J. & Czajkowski, C. Identification of transduction elements for benzodiazepine modulation of the GABAA receptor: three residues are required for allosteric coupling. J. Neurosci. 19, 10213–10220 (1999).

  40. 40

    Lynch, J.W. et al. Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. EMBO J. 16, 110–120 (1997).

  41. 41

    O'Shea, S.M. & Harrison, N.L. Arg274 and Leu277 of the γ-aminobutyric acid type A receptor α 2 subunit define agonist efficacy and potency. J. Biol. Chem. 275, 22764–22768 (2000).

  42. 42

    Fisher, J.L. & MacDonald, R.L. The role of an α subtype M2-M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations. J. Neurosci. 18, 2944–2953 (1998).

  43. 43

    Cromer, B.A., Morton, C.J. & Parker, M.W. Anxiety over GABAA receptor structure relieved by AChBP. Trends Biochem. Sci. 27, 280–287 (2002).

  44. 44

    Pawelzik, H., Bannister, A.P., Deuchars, J., Ilia, M. & Thomson, A.M. Modulation of bistratified cell IPSPs and basket cell IPSPs by pentobarbitone sodium, diazepam and Zn2+: dual recordings in slices of adult rat hippocampus. Eur. J. Neurosci. 11, 3552–3564 (1999).

  45. 45

    Wang, Z., Danscher, G., Kim, Y.K., Dahlstrom, A. & Mook Jo, S. Inhibitory zinc-enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase. Neurosci. Lett. 321, 37–40 (2002).

  46. 46

    Sieghart, W. & Sperk, G. Subunit composition, distribution and function of GABAA receptor subtypes. Curr. Top. Med. Chem. 2, 795–816 (2002).

  47. 47

    Brickley, S.G., Cull-Candy, S.G. & Farrant, M. Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. J. Neurosci. 19, 2960–2973 (1999).

  48. 48

    Taylor, P.M. et al. Identification of residues within GABAA receptor alpha subunits that mediate specific assembly with receptor beta subunits. J. Neurosci. 20, 1297–1306 (2000).

  49. 49

    Xu, M. & Akabas, M.H. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAA receptor alpha1 subunit. J. Gen. Physiol. 107, 195–205 (1996).

Download references

Acknowledgements

This work was supported by the Medical Research Council (UK). E.L.D. was a University of London Maplethorpe Research Fellow. We thank P. Thomas and P. Miller for helpful comments.

Author information

Competing interests

The authors declare no competing financial interests.

Correspondence to Trevor G. Smart.

Supplementary information

  1. Supplementary Fig. 1.

    Schematic model of Zn2+ inhibition on the GABAA receptor. The α1β3 and α1β3γ2 GABAA receptors are viewed as plans and also as transected sections that span the membrane. The plan view depicts the interfacial nature of the GABA, benzodiazepine (BZ) and Zn2+ binding sites where they are intact (black spheres) or disrupted (shaded spheres). In the transected views, the location of receptor subunit interfaces, the GABA binding site and the determinants of Zn2+ potency (shaded ovals) were derived by comparison with the AChBP30. The GABA and BZ binding sites, and the domains delineated by α1E122,D123, α1E137,H141, and β3E182, are located externally on the receptor (solid lines, shown also on the oblique side of the receptor as broken lines) in contrast to the channel mouth location of H267 and E270 in the β3 subunit. The three postulated types of Zn2+ binding site, formed by H267 and E270, and by E137, H141 and E182, act in a concerted, allosteric fashion to inhibit receptor function. The channel site will be severely disrupted by introduction of γ2 subunits. (PDF 503 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading

Figure 1: Zinc inhibition of GABA-activated currents is not solely reliant on His267 in the β subunit.
Figure 2: Scanning the α1 subunit for Zn2+ binding residues.
Figure 3: Zn2+ potency is affected by both external histidine and acidic residues.
Figure 4: Structural location of interface residues affecting Zn2+ inhibition.
Figure 5: Three clusters of residues underlie Zn2+ inhibition on α1β3 receptors.
Figure 6: The low Zn2+ sensitivity of αβγ receptors reflects the disruption of the ion channel and of an extracellular site.