Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Apr;78(4):1786–1803. doi: 10.1016/S0006-3495(00)76729-4

Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships.

B S Zhorov 1, P D Bregestovski 1
PMCID: PMC1300774  PMID: 10733960

Abstract

GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundström, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to alpha(1) GlyR and rho(1) GABAR, the rounded end of PTX accepts several H-bonds from Thr(6')s, while the elongated end enters ring R(2'). The lack of H-bond donors on the side chains of Phe(6')s (beta GlyR) and Met(6')s (rho(2) GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro(2')s/Ala(2')s in GABARs, but fit a more hydrophilic ring with Gly(2')s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abagyan R., Totrov M. Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J Mol Biol. 1994 Jan 21;235(3):983–1002. doi: 10.1006/jmbi.1994.1052. [DOI] [PubMed] [Google Scholar]
  2. Adcock C., Smith G. R., Sansom M. S. Electrostatics and the ion selectivity of ligand-gated channels. Biophys J. 1998 Sep;75(3):1211–1222. doi: 10.1016/S0006-3495(98)74040-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Akabas M. H., Karlin A. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. Biochemistry. 1995 Oct 3;34(39):12496–12500. doi: 10.1021/bi00039a002. [DOI] [PubMed] [Google Scholar]
  4. Akabas M. H., Kaufmann C., Archdeacon P., Karlin A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron. 1994 Oct;13(4):919–927. doi: 10.1016/0896-6273(94)90257-7. [DOI] [PubMed] [Google Scholar]
  5. Anthony N. M., Holyoke C. W., Jr, Sattelle D. B. Blocking actions of picrotoxinin analogues on insect (Periplaneta americana) GABA receptors. Neurosci Lett. 1994 Apr 25;171(1-2):67–69. doi: 10.1016/0304-3940(94)90606-8. [DOI] [PubMed] [Google Scholar]
  6. Aqvist J., Luecke H., Quiocho F. A., Warshel A. Dipoles localized at helix termini of proteins stabilize charges. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):2026–2030. doi: 10.1073/pnas.88.5.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arias H. R. Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor. Biochim Biophys Acta. 1998 Aug 21;1376(2):173–220. doi: 10.1016/s0304-4157(98)00004-5. [DOI] [PubMed] [Google Scholar]
  8. Betz H. Ligand-gated ion channels in the brain: the amino acid receptor superfamily. Neuron. 1990 Oct;5(4):383–392. doi: 10.1016/0896-6273(90)90077-s. [DOI] [PubMed] [Google Scholar]
  9. Bormann J., Hamill O. P., Sakmann B. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. J Physiol. 1987 Apr;385:243–286. doi: 10.1113/jphysiol.1987.sp016493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brovtsyna N. B., Tikhonov D. B., Gorbunova O. B., Gmiro V. E., Serduk S. E., Lukomskaya N. Y., Magazanik L. G., Zhorov B. S. Architecture of the neuronal nicotinic acetylcholine receptor ion channel at the binding site of bis-ammonium blockers. J Membr Biol. 1996 Jul;152(1):77–87. doi: 10.1007/s002329900087. [DOI] [PubMed] [Google Scholar]
  11. Chang G., Spencer R. H., Lee A. T., Barclay M. T., Rees D. C. Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science. 1998 Dec 18;282(5397):2220–2226. doi: 10.1126/science.282.5397.2220. [DOI] [PubMed] [Google Scholar]
  12. Changeux J. P., Galzi J. L., Devillers-Thiéry A., Bertrand D. The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis. Q Rev Biophys. 1992 Nov;25(4):395–432. doi: 10.1017/s0033583500004352. [DOI] [PubMed] [Google Scholar]
  13. Cleland T. A. Inhibitory glutamate receptor channels. Mol Neurobiol. 1996 Oct;13(2):97–136. doi: 10.1007/BF02740637. [DOI] [PubMed] [Google Scholar]
  14. Davis W. C., Ticku M. K. Picrotoxinin and diazepam bind to two distinct proteins: further evidence that pentobarbital may act at the picrotoxinin site. J Neurosci. 1981 Sep;1(9):1036–1042. doi: 10.1523/JNEUROSCI.01-09-01036.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  16. Dwyer T. M., Adams D. J., Hille B. The permeability of the endplate channel to organic cations in frog muscle. J Gen Physiol. 1980 May;75(5):469–492. doi: 10.1085/jgp.75.5.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Enna S. J., Collins J. F., Snyder S. H. Stereospecificity and structure--activity requirements of GABA receptor binding in rat brain. Brain Res. 1977 Mar 18;124(1):185–190. doi: 10.1016/0006-8993(77)90878-2. [DOI] [PubMed] [Google Scholar]
  18. Enz R., Bormann J. A single point mutation decreases picrotoxinin sensitivity of the human GABA receptor rho 1 subunit. Neuroreport. 1995 Jul 31;6(11):1569–1572. doi: 10.1097/00001756-199507310-00026. [DOI] [PubMed] [Google Scholar]
  19. Fucile S., de Saint Jan D., David-Watine B., Korn H., Bregestovski P. Comparison of glycine and GABA actions on the zebrafish homomeric glycine receptor. J Physiol. 1999 Jun 1;517(Pt 2):369–383. doi: 10.1111/j.1469-7793.1999.0369t.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Galzi J. L., Changeux J. P. Neuronal nicotinic receptors: molecular organization and regulations. Neuropharmacology. 1995 Jun;34(6):563–582. doi: 10.1016/0028-3908(95)00034-4. [DOI] [PubMed] [Google Scholar]
  21. Galzi J. L., Devillers-Thiéry A., Hussy N., Bertrand S., Changeux J. P., Bertrand D. Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature. 1992 Oct 8;359(6395):500–505. doi: 10.1038/359500a0. [DOI] [PubMed] [Google Scholar]
  22. Gurley D., Amin J., Ross P. C., Weiss D. S., White G. Point mutations in the M2 region of the alpha, beta, or gamma subunit of the GABAA channel that abolish block by picrotoxin. Receptors Channels. 1995;3(1):13–20. [PubMed] [Google Scholar]
  23. Gurley D., Amin J., Ross P. C., Weiss D. S., White G. Point mutations in the M2 region of the alpha, beta, or gamma subunit of the GABAA channel that abolish block by picrotoxin. Receptors Channels. 1995;3(1):13–20. [PubMed] [Google Scholar]
  24. Horenstein J., Akabas M. H. Location of a high affinity Zn2+ binding site in the channel of alpha1beta1 gamma-aminobutyric acidA receptors. Mol Pharmacol. 1998 May;53(5):870–877. [PubMed] [Google Scholar]
  25. Hosie A. M., Aronstein K., Sattelle D. B., ffrench-Constant R. H. Molecular biology of insect neuronal GABA receptors. Trends Neurosci. 1997 Dec;20(12):578–583. doi: 10.1016/s0166-2236(97)01127-2. [DOI] [PubMed] [Google Scholar]
  26. Hucho F., Oberthür W., Lottspeich F. The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits. FEBS Lett. 1986 Sep 1;205(1):137–142. doi: 10.1016/0014-5793(86)80881-x. [DOI] [PubMed] [Google Scholar]
  27. Imoto K., Busch C., Sakmann B., Mishina M., Konno T., Nakai J., Bujo H., Mori Y., Fukuda K., Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 1988 Oct 13;335(6191):645–648. doi: 10.1038/335645a0. [DOI] [PubMed] [Google Scholar]
  28. Jarboe C. H., Poerter L. A., Buckler R. T. Structural aspects of picrotoxinin action. J Med Chem. 1968 Jul;11(4):729–731. doi: 10.1021/jm00310a020. [DOI] [PubMed] [Google Scholar]
  29. Karlin A., Akabas M. H. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron. 1995 Dec;15(6):1231–1244. doi: 10.1016/0896-6273(95)90004-7. [DOI] [PubMed] [Google Scholar]
  30. Labarca C., Nowak M. W., Zhang H., Tang L., Deshpande P., Lester H. A. Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature. 1995 Aug 10;376(6540):514–516. doi: 10.1038/376514a0. [DOI] [PubMed] [Google Scholar]
  31. Langosch D., Thomas L., Betz H. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7394–7398. doi: 10.1073/pnas.85.19.7394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lester H. A. The permeation pathway of neurotransmitter-gated ion channels. Annu Rev Biophys Biomol Struct. 1992;21:267–292. doi: 10.1146/annurev.bb.21.060192.001411. [DOI] [PubMed] [Google Scholar]
  33. Lockhart D. J., Kim P. S. Electrostatic screening of charge and dipole interactions with the helix backbone. Science. 1993 Apr 9;260(5105):198–202. doi: 10.1126/science.8469972. [DOI] [PubMed] [Google Scholar]
  34. Lockhart D. J., Kim P. S. Internal stark effect measurement of the electric field at the amino terminus of an alpha helix. Science. 1992 Aug 14;257(5072):947–951. doi: 10.1126/science.1502559. [DOI] [PubMed] [Google Scholar]
  35. Lynch J. W., Rajendra S., Barry P. H., Schofield P. R. Mutations affecting the glycine receptor agonist transduction mechanism convert the competitive antagonist, picrotoxin, into an allosteric potentiator. J Biol Chem. 1995 Jun 9;270(23):13799–13806. doi: 10.1074/jbc.270.23.13799. [DOI] [PubMed] [Google Scholar]
  36. Magoski N. S., Bulloch A. G. Dopamine activates two different receptors to produce variability in sign at an identified synapse. J Neurophysiol. 1999 Mar;81(3):1330–1340. doi: 10.1152/jn.1999.81.3.1330. [DOI] [PubMed] [Google Scholar]
  37. Newland C. F., Cull-Candy S. G. On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol. 1992 Feb;447:191–213. doi: 10.1113/jphysiol.1992.sp018998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nutter T. J., Adams D. J. Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia. J Gen Physiol. 1995 Jun;105(6):701–723. doi: 10.1085/jgp.105.6.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ortells M. O., Lunt G. G. A mixed helix-beta-sheet model of the transmembrane region of the nicotinic acetylcholine receptor. Protein Eng. 1996 Jan;9(1):51–59. doi: 10.1093/protein/9.1.51. [DOI] [PubMed] [Google Scholar]
  40. Ortells M. O., Lunt G. G. Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci. 1995 Mar;18(3):121–127. doi: 10.1016/0166-2236(95)93887-4. [DOI] [PubMed] [Google Scholar]
  41. Pribilla I., Takagi T., Langosch D., Bormann J., Betz H. The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels. EMBO J. 1992 Dec;11(12):4305–4311. doi: 10.1002/j.1460-2075.1992.tb05529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Qian H., Dowling J. E. Pharmacology of novel GABA receptors found on rod horizontal cells of the white perch retina. J Neurosci. 1994 Jul;14(7):4299–4307. doi: 10.1523/JNEUROSCI.14-07-04299.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reddy G. L., Iwamoto T., Tomich J. M., Montal M. Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure. J Biol Chem. 1993 Jul 15;268(20):14608–14615. [PubMed] [Google Scholar]
  44. Revah F., Bertrand D., Galzi J. L., Devillers-Thiéry A., Mulle C., Hussy N., Bertrand S., Ballivet M., Changeux J. P. Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature. 1991 Oct 31;353(6347):846–849. doi: 10.1038/353846a0. [DOI] [PubMed] [Google Scholar]
  45. Rundström N., Schmieden V., Betz H., Bormann J., Langosch D. Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8950–8954. doi: 10.1073/pnas.91.19.8950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sankararamakrishnan R., Adcock C., Sansom M. S. The pore domain of the nicotinic acetylcholine receptor: molecular modeling, pore dimensions, and electrostatics. Biophys J. 1996 Oct;71(4):1659–1671. doi: 10.1016/S0006-3495(96)79370-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shirai Y., Hosie A. M., Buckingham S. D., Holyoke C. W., Jr, Baylis H. A., Sattelle D. B. Actions of picrotoxinin analogues on an expressed, homo-oligomeric GABA receptor of Drosophila melanogaster. Neurosci Lett. 1995 Apr 7;189(1):1–4. doi: 10.1016/0304-3940(95)11432-v. [DOI] [PubMed] [Google Scholar]
  48. Sitkoff D., Lockhart D. J., Sharp K. A., Honig B. Calculation of electrostatic effects at the amino terminus of an alpha helix. Biophys J. 1994 Dec;67(6):2251–2260. doi: 10.1016/S0006-3495(94)80709-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sukharev S. I., Blount P., Martinac B., Blattner F. R., Kung C. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature. 1994 Mar 17;368(6468):265–268. doi: 10.1038/368265a0. [DOI] [PubMed] [Google Scholar]
  50. Tikhonov D. B., Zhorov B. S. Kinked-helices model of the nicotinic acetylcholine receptor ion channel and its complexes with blockers: simulation by the Monte Carlo minimization method. Biophys J. 1998 Jan;74(1):242–255. doi: 10.1016/S0006-3495(98)77783-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Unwin N. Acetylcholine receptor channel imaged in the open state. Nature. 1995 Jan 5;373(6509):37–43. doi: 10.1038/373037a0. [DOI] [PubMed] [Google Scholar]
  52. Wang T. L., Guggino W. B., Cutting G. R. A novel gamma-aminobutyric acid receptor subunit (rho 2) cloned from human retina forms bicuculline-insensitive homooligomeric receptors in Xenopus oocytes. J Neurosci. 1994 Nov;14(11 Pt 1):6524–6531. doi: 10.1523/JNEUROSCI.14-11-06524.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang T. L., Hackam A. S., Guggino W. B., Cutting G. R. A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11751–11755. doi: 10.1073/pnas.92.25.11751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wilson G. G., Karlin A. The location of the gate in the acetylcholine receptor channel. Neuron. 1998 Jun;20(6):1269–1281. doi: 10.1016/s0896-6273(00)80506-1. [DOI] [PubMed] [Google Scholar]
  55. Xu M., Akabas M. H. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit. J Gen Physiol. 1996 Feb;107(2):195–205. doi: 10.1085/jgp.107.2.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Xu M., Covey D. F., Akabas M. H. Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants. Biophys J. 1995 Nov;69(5):1858–1867. doi: 10.1016/S0006-3495(95)80056-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yang J. Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells. J Gen Physiol. 1990 Dec;96(6):1177–1198. doi: 10.1085/jgp.96.6.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yarowsky J., Carpenter D. O. A comparison of similar ionic responses to gamma-aminobutyric acid and acetylcholine. J Neurophysiol. 1978 May;41(3):531–541. doi: 10.1152/jn.1978.41.3.531. [DOI] [PubMed] [Google Scholar]
  59. Yoon K. W., Covey D. F., Rothman S. M. Multiple mechanisms of picrotoxin block of GABA-induced currents in rat hippocampal neurons. J Physiol. 1993 May;464:423–439. doi: 10.1113/jphysiol.1993.sp019643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhang D., Pan Z. H., Zhang X., Brideau A. D., Lipton S. A. Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11756–11760. doi: 10.1073/pnas.92.25.11756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhang H. G., Lee H. J., Rocheleau T., ffrench-Constant R. H., Jackson M. B. Subunit composition determines picrotoxin and bicuculline sensitivity of Drosophila gamma-aminobutyric acid receptors. Mol Pharmacol. 1995 Nov;48(5):835–840. [PubMed] [Google Scholar]
  62. Zhorov B. S., Ananthanarayanan V. S. Conformational analysis of free and Ca(2+)-bound forms of verapamil and methoxyverapamil. J Biomol Struct Dyn. 1993 Dec;11(3):529–540. doi: 10.1080/07391102.1993.10508013. [DOI] [PubMed] [Google Scholar]
  63. Zhorov B. S., Ananthanarayanan V. S. Structural model of a synthetic Ca2+ channel with bound Ca2+ ions and dihydropyridine ligand. Biophys J. 1996 Jan;70(1):22–37. doi: 10.1016/S0006-3495(96)79561-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhorov B. S., Brovtsyna N. B., Gmiro V. E., Lukomskaya NYa, Serdyuk S. E., Potapyeva N. N., Magazanik L. G., Kurenniy D. E., Skok V. I. Dimensions of the ion channel in neuronal nicotinic acetylcholine receptor as estimated from analysis of conformation-activity relationships of open-channel blocking drugs. J Membr Biol. 1991 Apr;121(2):119–132. doi: 10.1007/BF01870527. [DOI] [PubMed] [Google Scholar]
  65. Zimmerman S. S., Pottle M. S., Némethy G., Scheraga H. A. Conformational analysis of the 20 naturally occurring amino acid residues using ECEPP. Macromolecules. 1977 Jan-Feb;10(1):1–9. doi: 10.1021/ma60055a001. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES