Abstract
Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations of residues five to seven positions downstream of the Q/R/N site do not influence the external Mg2+ block, but clearly influence the block by internal Mg2+. These data add support to the hypothesis that there are two separate binding sites for external and internal Mg2+ block. They also indicate that the C-terminal end of TMII contributes to the inner vestibule of the pore of NMDA channels and thus provide additional evidence that TMII forms a loop that reemerges toward the cytoplasmic side of the membrane.
Full text
PDF





Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arellano R. O., Woodward R. M., Miledi R. A monovalent cationic conductance that is blocked by extracellular divalent cations in Xenopus oocytes. J Physiol. 1995 May 1;484(Pt 3):593–604. doi: 10.1113/jphysiol.1995.sp020689. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ascher P., Nowak L. The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J Physiol. 1988 May;399:247–266. doi: 10.1113/jphysiol.1988.sp017078. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennett J. A., Dingledine R. Topology profile for a glutamate receptor: three transmembrane domains and a channel-lining reentrant membrane loop. Neuron. 1995 Feb;14(2):373–384. doi: 10.1016/0896-6273(95)90293-7. [DOI] [PubMed] [Google Scholar]
- Burnashev N., Schoepfer R., Monyer H., Ruppersberg J. P., Günther W., Seeburg P. H., Sakmann B. Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor. Science. 1992 Sep 4;257(5075):1415–1419. doi: 10.1126/science.1382314. [DOI] [PubMed] [Google Scholar]
- Dingledine R., Hume R. I., Heinemann S. F. Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels. J Neurosci. 1992 Oct;12(10):4080–4087. doi: 10.1523/JNEUROSCI.12-10-04080.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hollmann M., Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci. 1994;17:31–108. doi: 10.1146/annurev.ne.17.030194.000335. [DOI] [PubMed] [Google Scholar]
- Hollmann M., Maron C., Heinemann S. N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron. 1994 Dec;13(6):1331–1343. doi: 10.1016/0896-6273(94)90419-7. [DOI] [PubMed] [Google Scholar]
- Jahr C. E., Stevens C. F. Voltage dependence of NMDA-activated macroscopic conductances predicted by single-channel kinetics. J Neurosci. 1990 Sep;10(9):3178–3182. doi: 10.1523/JNEUROSCI.10-09-03178.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jobling S. A., Gehrke L. Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature. 1987 Feb 12;325(6105):622–625. doi: 10.1038/325622a0. [DOI] [PubMed] [Google Scholar]
- Johnson J. W., Ascher P. Equilibrium and kinetic study of glycine action on the N-methyl-D-aspartate receptor in cultured mouse brain neurons. J Physiol. 1992 Sep;455:339–365. doi: 10.1113/jphysiol.1992.sp019305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson J. W., Ascher P. Voltage-dependent block by intracellular Mg2+ of N-methyl-D-aspartate-activated channels. Biophys J. 1990 May;57(5):1085–1090. doi: 10.1016/S0006-3495(90)82626-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li-Smerin Y., Johnson J. W. Effects of intracellular Mg2+ on channel gating and steady-state responses of the NMDA receptor in cultured rat neurons. J Physiol. 1996 Feb 15;491(Pt 1):137–150. doi: 10.1113/jphysiol.1996.sp021202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li-Smerin Y., Johnson J. W. Kinetics of the block by intracellular Mg2+ of the NMDA-activated channel in cultured rat neurons. J Physiol. 1996 Feb 15;491(Pt 1):121–135. doi: 10.1113/jphysiol.1996.sp021201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lu Z., MacKinnon R. Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel. Nature. 1994 Sep 15;371(6494):243–246. doi: 10.1038/371243a0. [DOI] [PubMed] [Google Scholar]
- MacKinnon R., Latorre R., Miller C. Role of surface electrostatics in the operation of a high-conductance Ca2+-activated K+ channel. Biochemistry. 1989 Oct 3;28(20):8092–8099. doi: 10.1021/bi00446a020. [DOI] [PubMed] [Google Scholar]
- Mayer M. L., Westbrook G. L., Guthrie P. B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature. 1984 May 17;309(5965):261–263. doi: 10.1038/309261a0. [DOI] [PubMed] [Google Scholar]
- Mayer M. L., Westbrook G. L. Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones. J Physiol. 1987 Dec;394:501–527. doi: 10.1113/jphysiol.1987.sp016883. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McBain C. J., Mayer M. L. N-methyl-D-aspartic acid receptor structure and function. Physiol Rev. 1994 Jul;74(3):723–760. doi: 10.1152/physrev.1994.74.3.723. [DOI] [PubMed] [Google Scholar]
- McLaughlin S. The electrostatic properties of membranes. Annu Rev Biophys Biophys Chem. 1989;18:113–136. doi: 10.1146/annurev.bb.18.060189.000553. [DOI] [PubMed] [Google Scholar]
- Miledi R. A calcium-dependent transient outward current in Xenopus laevis oocytes. Proc R Soc Lond B Biol Sci. 1982 Jul 22;215(1201):491–497. doi: 10.1098/rspb.1982.0056. [DOI] [PubMed] [Google Scholar]
- Monyer H., Sprengel R., Schoepfer R., Herb A., Higuchi M., Lomeli H., Burnashev N., Sakmann B., Seeburg P. H. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science. 1992 May 22;256(5060):1217–1221. doi: 10.1126/science.256.5060.1217. [DOI] [PubMed] [Google Scholar]
- Mori H., Masaki H., Yamakura T., Mishina M. Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature. 1992 Aug 20;358(6388):673–675. doi: 10.1038/358673a0. [DOI] [PubMed] [Google Scholar]
- Moriyoshi K., Masu M., Ishii T., Shigemoto R., Mizuno N., Nakanishi S. Molecular cloning and characterization of the rat NMDA receptor. Nature. 1991 Nov 7;354(6348):31–37. doi: 10.1038/354031a0. [DOI] [PubMed] [Google Scholar]
- Nowak L., Bregestovski P., Ascher P., Herbet A., Prochiantz A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature. 1984 Feb 2;307(5950):462–465. doi: 10.1038/307462a0. [DOI] [PubMed] [Google Scholar]
- Paoletti P., Neyton J., Ascher P. Glycine-independent and subunit-specific potentiation of NMDA responses by extracellular Mg2+. Neuron. 1995 Nov;15(5):1109–1120. doi: 10.1016/0896-6273(95)90099-3. [DOI] [PubMed] [Google Scholar]
- Sakurada K., Masu M., Nakanishi S. Alteration of Ca2+ permeability and sensitivity to Mg2+ and channel blockers by a single amino acid substitution in the N-methyl-D-aspartate receptor. J Biol Chem. 1993 Jan 5;268(1):410–415. [PubMed] [Google Scholar]
- Schoenmakers T. J., Visser G. J., Flik G., Theuvenet A. P. CHELATOR: an improved method for computing metal ion concentrations in physiological solutions. Biotechniques. 1992 Jun;12(6):870-4, 876-9. [PubMed] [Google Scholar]
- Stern-Bach Y., Bettler B., Hartley M., Sheppard P. O., O'Hara P. J., Heinemann S. F. Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron. 1994 Dec;13(6):1345–1357. doi: 10.1016/0896-6273(94)90420-0. [DOI] [PubMed] [Google Scholar]
- Wo Z. G., Oswald R. E. Transmembrane topology of two kainate receptor subunits revealed by N-glycosylation. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7154–7158. doi: 10.1073/pnas.91.15.7154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woodhull A. M. Ionic blockage of sodium channels in nerve. J Gen Physiol. 1973 Jun;61(6):687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yellen G., Jurman M. E., Abramson T., MacKinnon R. Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel. Science. 1991 Feb 22;251(4996):939–942. doi: 10.1126/science.2000494. [DOI] [PubMed] [Google Scholar]
- Zarei M. M., Dani J. A. Structural basis for explaining open-channel blockade of the NMDA receptor. J Neurosci. 1995 Feb;15(2):1446–1454. doi: 10.1523/JNEUROSCI.15-02-01446.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]