Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Jul;83(1):252–262. doi: 10.1016/S0006-3495(02)75166-7

NMR structures of the second transmembrane domain of the human glycine receptor alpha(1) subunit: model of pore architecture and channel gating.

Pei Tang 1, Pravat K Mandal 1, Yan Xu 1
PMCID: PMC1302144  PMID: 12080117

Abstract

Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR alpha(1) subunit are unique in that they form functional homopentameric channels with conductance characteristics nearly identical to those of an authentic receptor (Opella, S. J., J. Gesell, A. R. Valente, F. M. Marassi, M. Oblatt-Montal, W. Sun, A. F. Montiel, and M. Montal. 1997. Chemtracts Biochem. Mol. Biol. 10:153-174). Using NMR and circular dichroism (CD), we determined the high-resolution structures of the TM2 segment of human alpha(1) GlyR and an anesthetic-insensitive mutant (S267Y) in dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. The NMR structures showed right-handed alpha-helices without kinks. A well-defined hydrophilic path, composed of side chains of G2', T6', T10', Q14', and S18', runs along the helical surfaces at an angle approximately 10-20 degrees relative to the long axis of the helices. The side-chain arrangement of the NMR-derived structures and the energy minimization of a homopentameric TM2 channel in a fully hydrated DMPC membrane using large-scale computation suggest a model of pore architecture in which simultaneous tilting movements of entire TM2 helices by a mere 10 degrees may be sufficient to account for the channel gating. The model also suggests that additional residues accessible from within the pore include L3', T7', T13', and G17'. A similar pore architecture and gating mechanism may apply to other channels in the same superfamily, including GABA(A), nACh, and 5-HT(3) receptors.

Full Text

The Full Text of this article is available as a PDF (348.5 KB).

Selected References

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

  1. 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]
  2. Brejc K., van Dijk W. J., Klaassen R. V., Schuurmans M., van Der Oost J., Smit A. B., Sixma T. K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 2001 May 17;411(6835):269–276. doi: 10.1038/35077011. [DOI] [PubMed] [Google Scholar]
  3. Choma C., Gratkowski H., Lear J. D., DeGrado W. F. Asparagine-mediated self-association of a model transmembrane helix. Nat Struct Biol. 2000 Feb;7(2):161–166. doi: 10.1038/72440. [DOI] [PubMed] [Google Scholar]
  4. Delaglio F., Grzesiek S., Vuister G. W., Zhu G., Pfeifer J., Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995 Nov;6(3):277–293. doi: 10.1007/BF00197809. [DOI] [PubMed] [Google Scholar]
  5. Elmslie F. V., Hutchings S. M., Spencer V., Curtis A., Covanis T., Gardiner R. M., Rees M. Analysis of GLRA1 in hereditary and sporadic hyperekplexia: a novel mutation in a family cosegregating for hyperekplexia and spastic paraparesis. J Med Genet. 1996 May;33(5):435–436. doi: 10.1136/jmg.33.5.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fatima-Shad K., Barry P. H. Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons. Proc Biol Sci. 1993 Jul 22;253(1336):69–75. doi: 10.1098/rspb.1993.0083. [DOI] [PubMed] [Google Scholar]
  7. Franks N. P., Lieb W. R. An anesthetic-sensitive superfamily of neurotransmitter-gated ion channels. J Clin Anesth. 1996 May;8(3 Suppl):3S–7S. doi: 10.1016/s0952-8180(96)90004-5. [DOI] [PubMed] [Google Scholar]
  8. Grenningloh G., Schmieden V., Schofield P. R., Seeburg P. H., Siddique T., Mohandas T. K., Becker C. M., Betz H. Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes. EMBO J. 1990 Mar;9(3):771–776. doi: 10.1002/j.1460-2075.1990.tb08172.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996 Feb;14(1):33-8, 27-8. doi: 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]
  11. Karlin A., Akabas M. H. Substituted-cysteine accessibility method. Methods Enzymol. 1998;293:123–145. doi: 10.1016/s0076-6879(98)93011-7. [DOI] [PubMed] [Google Scholar]
  12. Killian J. A., Trouard T. P., Greathouse D. V., Chupin V., Lindblom G. A general method for the preparation of mixed micelles of hydrophobic peptides and sodium dodecyl sulphate. FEBS Lett. 1994 Jul 11;348(2):161–165. doi: 10.1016/0014-5793(94)00594-x. [DOI] [PubMed] [Google Scholar]
  13. Kochendoerfer G. G., Salom D., Lear J. D., Wilk-Orescan R., Kent S. B., DeGrado W. F. Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly. Biochemistry. 1999 Sep 14;38(37):11905–11913. doi: 10.1021/bi990720m. [DOI] [PubMed] [Google Scholar]
  14. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  15. Laskowski R. A., Rullmannn J. A., MacArthur M. W., Kaptein R., Thornton J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR. 1996 Dec;8(4):477–486. doi: 10.1007/BF00228148. [DOI] [PubMed] [Google Scholar]
  16. Lewis T. M., Sivilotti L. G., Colquhoun D., Gardiner R. M., Schoepfer R., Rees M. Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes. J Physiol. 1998 Feb 15;507(Pt 1):25–40. doi: 10.1111/j.1469-7793.1998.025bu.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li R., Babu C. R., Lear J. D., Wand A. J., Bennett J. S., DeGrado W. F. Oligomerization of the integrin alphaIIbbeta3: roles of the transmembrane and cytoplasmic domains. Proc Natl Acad Sci U S A. 2001 Oct 16;98(22):12462–12467. doi: 10.1073/pnas.221463098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Luo P., Baldwin R. L. Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemistry. 1997 Jul 8;36(27):8413–8421. doi: 10.1021/bi9707133. [DOI] [PubMed] [Google Scholar]
  19. Lynch J. W., Jacques P., Pierce K. D., Schofield P. R. Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways. J Neurochem. 1998 Nov;71(5):2159–2168. doi: 10.1046/j.1471-4159.1998.71052159.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lynch J. W., Rajendra S., Pierce K. D., Handford C. A., Barry P. H., Schofield P. R. Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. EMBO J. 1997 Jan 2;16(1):110–120. doi: 10.1093/emboj/16.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  23. Marsh D. Peptide models for membrane channels. Biochem J. 1996 Apr 15;315(Pt 2):345–361. doi: 10.1042/bj3150345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mascia M. P., Trudell J. R., Harris R. A. Specific binding sites for alcohols and anesthetics on ligand-gated ion channels. Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9305–9310. doi: 10.1073/pnas.160128797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mihic S. J., Ye Q., Wick M. J., Koltchine V. V., Krasowski M. D., Finn S. E., Mascia M. P., Valenzuela C. F., Hanson K. K., Greenblatt E. P. Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature. 1997 Sep 25;389(6649):385–389. doi: 10.1038/38738. [DOI] [PubMed] [Google Scholar]
  26. Miller C. Genetic manipulation of ion channels: a new approach to structure and mechanism. Neuron. 1989 Mar;2(3):1195–1205. doi: 10.1016/0896-6273(89)90304-8. [DOI] [PubMed] [Google Scholar]
  27. Moorhouse A. J., Jacques P., Barry P. H., Schofield P. R. The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating. Mol Pharmacol. 1999 Feb;55(2):386–395. doi: 10.1124/mol.55.2.386. [DOI] [PubMed] [Google Scholar]
  28. Nilges M., Clore G. M., Gronenborn A. M. Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. Circumventing problems associated with folding. FEBS Lett. 1988 Oct 24;239(1):129–136. doi: 10.1016/0014-5793(88)80559-3. [DOI] [PubMed] [Google Scholar]
  29. Opella S. J., Marassi F. M., Gesell J. J., Valente A. P., Kim Y., Oblatt-Montal M., Montal M. Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy. Nat Struct Biol. 1999 Apr;6(4):374–379. doi: 10.1038/7610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Piotto M., Saudek V., Sklenár V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992 Nov;2(6):661–665. doi: 10.1007/BF02192855. [DOI] [PubMed] [Google Scholar]
  31. Rajendra S., Lynch J. W., Pierce K. D., French C. R., Barry P. H., Schofield P. R. Mutation of an arginine residue in the human glycine receptor transforms beta-alanine and taurine from agonists into competitive antagonists. Neuron. 1995 Jan;14(1):169–175. doi: 10.1016/0896-6273(95)90251-1. [DOI] [PubMed] [Google Scholar]
  32. Rajendra S., Lynch J. W., Schofield P. R. The glycine receptor. Pharmacol Ther. 1997;73(2):121–146. doi: 10.1016/s0163-7258(96)00163-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Rees M. I., Andrew M., Jawad S., Owen M. J. Evidence for recessive as well as dominant forms of startle disease (hyperekplexia) caused by mutations in the alpha 1 subunit of the inhibitory glycine receptor. Hum Mol Genet. 1994 Dec;3(12):2175–2179. doi: 10.1093/hmg/3.12.2175. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Salom D., Hill B. R., Lear J. D., DeGrado W. F. pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus. Biochemistry. 2000 Nov 21;39(46):14160–14170. doi: 10.1021/bi001799u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scholtz J. M., Barrick D., York E. J., Stewart J. M., Baldwin R. L. Urea unfolding of peptide helices as a model for interpreting protein unfolding. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):185–189. doi: 10.1073/pnas.92.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shiang R., Ryan S. G., Zhu Y. Z., Fielder T. J., Allen R. J., Fryer A., Yamashita S., O'Connell P., Wasmuth J. J. Mutational analysis of familial and sporadic hyperekplexia. Ann Neurol. 1995 Jul;38(1):85–91. doi: 10.1002/ana.410380115. [DOI] [PubMed] [Google Scholar]
  39. Shiang R., Ryan S. G., Zhu Y. Z., Hahn A. F., O'Connell P., Wasmuth J. J. Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat Genet. 1993 Dec;5(4):351–358. doi: 10.1038/ng1293-351. [DOI] [PubMed] [Google Scholar]
  40. Smart O. S., Neduvelil J. G., Wang X., Wallace B. A., Sansom M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J Mol Graph. 1996 Dec;14(6):354-60, 376. doi: 10.1016/s0263-7855(97)00009-x. [DOI] [PubMed] [Google Scholar]
  41. Tang P., Eckenhoff R. G., Xu Y. General anesthetic binding to gramicidin A: the structural requirements. Biophys J. 2000 Apr;78(4):1804–1809. doi: 10.1016/S0006-3495(00)76730-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tang P., Hu J., Liachenko S., Xu Y. Distinctly different interactions of anesthetic and nonimmobilizer with transmembrane channel peptides. Biophys J. 1999 Aug;77(2):739–746. doi: 10.1016/S0006-3495(99)76928-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tang P., Simplaceanu V., Xu Y. Structural consequences of anesthetic and nonimmobilizer interaction with gramicidin A channels. Biophys J. 1999 May;76(5):2346–2350. doi: 10.1016/S0006-3495(99)77391-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tang P., Yan B., Xu Y. Different distribution of fluorinated anesthetics and nonanesthetics in model membrane: a 19F NMR study. Biophys J. 1997 Apr;72(4):1676–1682. doi: 10.1016/S0006-3495(97)78813-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tatulian S. A., Tamm L. K. Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin. Biochemistry. 2000 Jan 25;39(3):496–507. doi: 10.1021/bi991594p. [DOI] [PubMed] [Google Scholar]
  46. Tochio H., Ohki S., Zhang Q., Li M., Zhang M. Solution structure of a protein inhibitor of neuronal nitric oxide synthase. Nat Struct Biol. 1998 Nov;5(11):965–969. doi: 10.1038/2940. [DOI] [PubMed] [Google Scholar]
  47. Twyman R. E., Macdonald R. L. Kinetic properties of the glycine receptor main- and sub-conductance states of mouse spinal cord neurones in culture. J Physiol. 1991 Apr;435:303–331. doi: 10.1113/jphysiol.1991.sp018512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Unwin N. The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission. Philos Trans R Soc Lond B Biol Sci. 2000 Dec 29;355(1404):1813–1829. doi: 10.1098/rstb.2000.0737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Unwin N. The nicotinic acetylcholine receptor of the Torpedo electric ray. J Struct Biol. 1998;121(2):181–190. doi: 10.1006/jsbi.1997.3949. [DOI] [PubMed] [Google Scholar]
  51. Wilson G., Karlin A. Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method. Proc Natl Acad Sci U S A. 2001 Jan 16;98(3):1241–1248. doi: 10.1073/pnas.031567798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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 beta subunits. J Physiol. 1997 Dec 15;505(Pt 3):633–640. doi: 10.1111/j.1469-7793.1997.633ba.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Xu Y., Tang P. Amphiphilic sites for general anesthetic action? Evidence from 129Xe-[1H] intermolecular nuclear Overhauser effects. Biochim Biophys Acta. 1997 Jan 14;1323(1):154–162. doi: 10.1016/s0005-2736(96)00184-8. [DOI] [PubMed] [Google Scholar]
  56. Xu Y., Tang P., Liachenko S. Unifying characteristics of sites of anesthetic action revealed by combined use of anesthetics and non-anesthetics. Toxicol Lett. 1998 Nov 23;100-101:347–352. doi: 10.1016/s0378-4274(98)00205-7. [DOI] [PubMed] [Google Scholar]
  57. Zhou F. X., Cocco M. J., Russ W. P., Brunger A. T., Engelman D. M. Interhelical hydrogen bonding drives strong interactions in membrane proteins. Nat Struct Biol. 2000 Feb;7(2):154–160. doi: 10.1038/72430. [DOI] [PubMed] [Google Scholar]

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

RESOURCES