Abstract
Glutamate receptors are large integral membrane proteins that belong to the class of ligand-gated ion channels. They constitute a family of receptors that are activated by binding of the neurotransmitter l-glutamate. Functional receptors are formed by assembly of several subunits around a central ion-conducting pore. Each subunit is thought to contain multiple membrane-spanning domains. A reliable membrane topology model, which defines the number of transmembrane segments and their orientation, is critical for understanding the relationship between the structure of these receptors and their function. This chapter describes some of the technical considerations of an approach that we and others have used to determine experimentally aspects of the membrane topology of ionotropic glutamate receptors (1–7). This strategy uses the fact that glycosylation of asparagine residues only occurs in extracellular domains, owing to the fact that the glycosyl transferases are localized to the lumen of the endoplasmatic reticulum. Demonstration of functional N-linked glycosylation therefore provides a definitive measure of extracellular localization. Alternative approaches that do not rely on alteration of N-glycosylation status have also provided information on membrane topology of glutamate receptors (7–9), but a detailed discussion of these methods is outside the scope of this chapter.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Taverna, F. A., Wang, L. Y., MacDonald, J. F., and Hampson, D. R. (1994) A transmembrane model for an ionotropic glutamate receptor predicted on the basis of the location of asparagine-linked oligosaccharides. J. Biol. Chem. 269, 14,159–14,164.
Roche, K. W., Raymond, L. A., Blackstone, C., and Huganir, R. L. (1994) Transmembrane topology of the glutamate receptor subunit GluR6. J. Biol. Chem. 269, 11,679–11,682.
Wo, Z. G. and Oswald, R. E. (1994) Transmembrane topology of two kainate receptor subunits revealed by N-glycosylation. Proc. Natl. Acad. Sci. USA 91, 7154–7158.
Hollmann, M., Maron, C., and Heinemann, S. F. (1994) N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron 13, 1331–1343.
Wood, M. W., VanDongen, H. M. A., and VanDongen, A. M. J. (1995) Structural conservation of ion conduction pathways in K channels and glutamate receptors. Proc. Natl. Acad. Sci. USA 92, 4882–4886.
Wo, Z. G. and Oswald, R. E. (1995) A topological analysis of goldfish kainate receptors predicts three transmembrane segments. J. Biol. Chem. 270, 2000–2009.
Wood, M. W., VanDongen, H. M. A., and VanDongen, A. M. J. (1997) An alanine residue in the M3–M4 linker lines the glycine binding pocket of the N-Methyl-d-Aspartate receptor. J. Biol. Chem. 272, 3532–3537.
Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P. O., O’Hara, P. J., and Heinemann, S. F. (1994) Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron 13, 1345–1357.
Bennett, J. A. and Dingledine, R. (1995) Topology profile for a glutamate receptor: three transmembrane domains and a channel-ling re-entrant membrane loop. Neuron 14, 373–384.
Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132.
Gafvelin, G., Sakaguchi, M., Andersson, H., and Von Heijne, G. (1997) Topological rules for membrane protein assembly in eukaryotic cells. J. Biol. Chem. 272, 6119–6127.
Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., et al. (1982) Primary structure of alpha-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature 299, 793–797.
Akabas, M. H., Kaufmann, C., Archdeacon, P., and Karlin, A. (1994) Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron 13, 919–927.
Kuner, T., Wollmuth, L. P., Karlin, A., Seeburg, P. H., and Sakmann, B. (1996) Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines. Neuron 17, 343–352.
Pedersen, S. E., Bridgman, P. C., Sharp, S. D., and Cohen, J. B. (1990) Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor α-subunit by epitope mapping. J. Biol. Chem. 265, 569–581.
Ratnam, M., Nguyen, D. L., Rivier, J., Sargent, P. B., and Lindstrom, J. (1986) Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles. Biochemistry 25, 2633–2643.
Criado, M., Hochschwender, S., Sarin, V., Fox, J. L., and Lindstrom, J. (1985) Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits. Proc. Natl. Acad. Sci. USA 82, 2004–2008.
Wilkinson, B. M., Critchley, A. J., and Stirling, C. J. (1996) Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex. J. Biol. Chem. 271, 25,590–25,597.
Wang, C. C., Schultz, D. E., and Nicholas, R. A. (1996) Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli. Biochem. J. 316, 149–156.
Zen, K. H., Consler, T. G., and Kaback, H. R. (1995) Insertion of the polytopic membrane protein lactose permease occurs by multiple mechanisms. Biochemistry 34, 3430–3437.
Huganir, R. L. and Miles, K. (1989) Protein phosphorylation of nicotinic acetylcholine receptors. Crit. Rev. Biochem. Mol. Biol. 24, 183–215.
Tingley, W. G., Roche, K. W., Thompson, A. K., and Huganir, R. L. (1993) Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain. Nature 364, 70–73.
McGlade-McCulloh, E., Yamamoto, H., Tan, S., Brickey, D., and Soderling, T. (1993) Phosphorylation and regulation of glutamate receptors by calcium/calmodulin protein kinase II. Nature 362, 640–642.
Wang, L.-Y., Taverna, F. A., Huang, X.-P., MacDonald, J. F., and Hampson, D. R. (1993) Phosphorylation and modulation of a kainate receptor (GluR6) by cAMP-dependent protein kinase. Science 259, 1173–1175.
Nakazawa, K., Mikawa, S., Hashikawa, T., and Ito, M. (1995) Transient and persistent phosphorylation of AMPA-type glutamate receptor subunits in cerebellar Purkinje cells. Neuron 15, 697–709.
Yakel, J., Vissavajjhala, P., Derkach, V., Brickey, D., and Soderling, T. (1995) Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-d-aspartate glutamate receptors. Proc. Natl. Acad. Sci. USA 92, 1376–1380.
Colquhoun, D. and Farrant, M. (1993) Molecular pharmacology. The binding issue. Nature 366, 510–511.
Landolt-Martcorena, C. and Reithmeier, R. A. F. (1994) Asparagine-linked oligosacharides are localized to single extracytosolic segments in multi-span membrane glycoproteins. Biochem. J. 302, 253–260.
Gafvelin, G. and Von Heijne, G. (1994) Topological “frustration” in multispanning E. coli inner membrane proteins. Cell 77, 401–412.
Higgins, J. B. and Casey, P. J. (1994) In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex. J. Biol. Chem. 269, 9067–9073.
Wiley, H. S. and Wallace, R. A. (1981) The structure of vitellogenin. Multiple vitellogenins in Xenopus eggs give rise to multiple forms of the yolk protein. J. Biol. Chem. 256, 8626–8634.
Evans, J. P. and Kay, B. K. (1991) Biochemical fractionations of oocytes. Methods Cell Biol. 36, 133–148.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
VanDongen, H.M.A., VanDongen, A.M.J. (1999). Determination of Membrane Topology of Glutamate Receptors. In: Li, M. (eds) NMDA Receptor Protocols. Methods in Molecular Biology™, vol 128. Humana Press. https://doi.org/10.1385/1-59259-683-5:155
Download citation
DOI: https://doi.org/10.1385/1-59259-683-5:155
Publisher Name: Humana Press
Print ISBN: 978-0-89603-713-7
Online ISBN: 978-1-59259-683-6
eBook Packages: Springer Protocols