Intracellular Domains of NR 2 Alter Calcium-Dependent Inactivation of N-Methyl-D-aspartate Receptors

At central excitatory synapses, the transient elevation of intracellular calcium reduces N-methyl-D-aspartate (NMDA) receptor activity. Such ‘calcium-dependent inactivation’ is mediated by interactions of calcium/calmodulin and -actinin with the C terminus of NMDA receptor 1 (NR1) subunit. However, inactivation is also NR2-subunit specific, because it occurs in NR2Abut not NR2Ccontaining receptors. We examined the molecular basis for NR2subunit specificity using chimeric and mutated NMDA receptor subunits expressed in HEK293 cells. We report that the intracellular loop immediately distal to the pore-forming P-loop M2 (M2–3 loop), as well as a short region in the C terminus, are involved in NR2-subunit specificity. Within the M2–3 loop, substitution of residue 619 in NR2A (valine) for the corresponding NR2C residue (isoleucine) reduced inactivation without affecting calcium permeability of the channel. In contrast, a Q620E mutation in NR2A reduced the relative calcium permeability without altering inactivation. Mutation of three serine/threonine residues in the M2–3 loop also reduced inactivation, as did substitution of the intracellular C terminus of NR2A for NR2C. We speculate that the M2–3 loop of NR2 modulates calcium-dependent inactivation by interacting with the NR1 C terminus, a region known to be essential for inactivation. NMDA receptors are heteromers of NMDA receptor 1 (NR1), NR2, and, in some cases, NR3 subunits. Several characteristics of native NMDA channels, including magnesium sensitivity, glycine affinity, and desensitization, depend on both NR1 and NR2 (for review see McBain and Mayer, 1994; Dingledine et al., 1999). The molecular mechanisms responsible for NR2-subunit specific differences in receptor function are complex and not well characterized. For example, block by magnesium ions is largely determined by residues in the NR2 pore domain (M2) (Burnashev et al., 1992; Mori et al., 1992; Sakurada et al., 1993; Williams et al., 1998; Wollmuth et al., 1998), but regions outside of M2 are modulatory (Kuner and Schoepfer, 1996). Likewise, calcium-dependent inactivation (Legendre et al., 1993; Rosenmund and Westbrook, 1993) also depends on NR1 and NR2. Whereas the interactions of two intracellular proteins, calcium/calmodulin (Ehlers et al., 1996) and -actinin (Wyszynski et al., 1997), with the C-terminal region of NR1 are necessary for inactivation (Zhang et al., 1998; Krupp et al., 1999), the coexpressed NR2 subunit is permissive (Krupp et al., 1996). This effect of the NR2-subunit is not caused by the small differences in the calcium permeability between the different NR1/ NR2 heteromers (Krupp et al., 1996), suggesting that domains within NR2 influence inactivation. To determine domains in NR2 that influence inactivation, we expressed NR1/NR2 heteromers in HEK293 cells. We found that the valine residue at position 619 in the M2–3 loop of NR2A is critical for NR2 subunit specificity. A point mutation that switched this residue in NR2A (valine) for NR2C (isoleucine) reduced inactivation without affecting calcium permeability. Mutagenesis of serine/threonine residues in the M2–3 loop of NR2 also affected inactivation. In addition, the proximal portion of the intracellular NR2 C terminus had a small modulatory effect. Materials and Methods Molecular Biology. The following NMDA subunit cDNAs were used: NR1–1a (GenBank accession number U08261), NR1–4a (GenBank accession number U08267) (Hollmann et al., 1993), NR2A (GenBank accession number D13211), and NR2C (GenBank accession number D13212) (Ishii et al., 1993). Most NR2A/2C chimeras have been described previously (Krupp et al., 1998), except for 2C 2 i A (NR2CM1–I630AQ620–V1464), 2C2 iiA (NR2CM1–E631AN621–V1464), and 2C3A (NR2CM1–L643AV633–V1464). Chimeras and mutants were generated using the strategy of gene splicing by overlap extension polymerase chain reaction (Horten et al., 1989) using Pfu polymerase (Stratagene, La Jolla, CA). All NMDA subunit cDNAs and chimeras This work was supported by National Institutes of Health grants MH46613 (G.L.W.), NS28709 (S.F.H.), the McKnight Foundation (S.F.H.), the John Adler Foundation (S.F.H.), fellowships from the Human Frontiers program (J.J.K. and B.V.), a Bundy Foundation award (B.V.) and a CJ Martin NHMRC of Australia award (B.V.). B.V. and J.J.K. contributed equally to this work. ABBREVIATIONS: NMDA, N-methyl-D-aspartate; NR, N-methyl-D-aspartate receptor; HEK, human embryonic kidney; Mx, membrane region, where x is 1, 2, or 3; DRB, 5,6-dichloro-1-D-ribofuranosylbenzimidazole. 0026-895X/02/6103-595–605$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 3 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1330/966217 Mol Pharmacol 61:595–605, 2002 Printed in U.S.A. 595 at A PE T Jornals on Jne 3, 2017 m oharm . aspeurnals.org D ow nladed from were cloned into pCDNA1/amp (Invitrogen, Carlsbad, CA). Truncation mutants were generated by introduction of a stop codon at the appropriate position. Amino acid numbers for mutations in wild-type subunits are as given in Ishii et al. (1993). For mutations in chimeras, we used the amino acid numbering of the embedding wild-type subunit. All clones were confirmed by restriction analysis and sequence analysis. To identify cells expressing NMDA receptors, HEK293 cells were cotransfected with cDNA coding for the lymphocyte CD4 receptor. The CD4 cDNA, kindly provided by Dr. John Adelman (Vollum Institute, Portland, OR), was inserted into the JPA vector. For detection of successfully transfected cells, 1 l of Dynabeads M-450 CD4 (Dynal, Norway) was added in 1 ml of medium to each 35-mm dish. The dish was then gently swirled for 15 to 20 min