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Interactions of Calmodulin and -Actinin with the NR1 Subunit Modulate Ca2 -Dependent Inactivation of NMDA Receptors

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Interactions of Calmodulin and -Actinin with the NR1 Subunit Modulate Ca2 -Dependent Inactivation of NMDA Receptors The Journal of Neuroscience, February 15, 1999, 19(4):1165–1178 Interactions of Calmodulin and a-Actinin with the NR1 Subunit Modulate Ca21-Dependent Inactivation of NMDA Receptors Johannes J. Krupp,1 Bryce Vissel,2 Christopher G. Thomas,1 Stephen F. Heinemann,2 and Gary L. Westbrook1 1Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, and 2Molecular Neurobiology Laboratory, Salk Institute, La Jolla, California 92037 Glutamate receptors are associated with various regulatory and in whole-cell recording, suggesting that inactivation in the intact cytoskeletal proteins. However, an understanding of the func- cell is more complex than binding of calmodulin to C0. Over- tional significance of these interactions is still rudimentary. expression of putative Ca 21-insensitive, but not Ca 21- Studies in hippocampal neurons suggest that such interactions sensitive, forms of a-actinin reduced inactivation, an effect that may be involved in calcium-induced reduction in the open was overcome by inclusion of calmodulin in the whole-cell probability of NMDA receptors (inactivation). Thus we examined pipette. The C0 domain also directly affects channel gating the role of the intracellular domains of the NR1 subunit and two because NR1 subunits with truncated C0 domains that lacked of its binding partners, calmodulin and a-actinin, on this pro- calmodulin or a-actinin binding sites had a low open probabil- cess using NR1/NR2A heteromers expressed in human embry- ity. We propose that inactivation can occur after C0 dissociates onic kidney (HEK) 293 cells. The presence of the first 30 resi- from a-actinin by two distinct but converging calcium- dues of the intracellular C terminus of NR1 (C0 domain) was dependent processes: competitive displacement of a-actinin required for inactivation. Mutations in the last five residues of by calmodulin and reduction in the affinity of a-actinin for C0 C0 reduced inactivation and produced parallel shifts in binding after binding of calcium to a-actinin. of a-actinin and Ca 21/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in ex- Key words: Ca/calmodulin; a-actinin; NR1 subunit; NMDA cised patches, calmodulin inhibitors did not block inactivation channel gating; open probability; protein–protein interactions Glutamate receptors at central synapses are clustered with a rich potential for protein–protein interactions, the influence of complex of proteins in the postsynaptic density (PSD). This these interactions on glutamate channel gating is relatively localization presumably is achieved by the association of recep- unknown. tors with other proteins in the PSD. Recent studies have led to the Calcium regulation of NMDA receptors has been postulated to hypothesis that cytoskeletal and regulatory proteins are included involve a linkage between the receptor and the cytoskeleton in this complex by binding to scaffolding proteins (Sheng and (Rosenmund and Westbrook, 1993b) and thus may provide a Wyszynski, 1997; O’Brien et al., 1998). In this view, scaffolding useful model for exploring interactions between membrane re- proteins not only localize and cluster receptors, but also facilitate ceptors and proteins in the subsynaptic scaffold. Inactivation the transduction of extracellular signals to specific intracellular results from a reduction in the open probability of the NMDA signal cascades. Consistent with differential localization of channel after elevation of intracellular calcium (Legendre et al., NMDA, AMPA, and metabotropic glutamate receptors, each 1993). Although the molecular mechanisms are not well under- class of receptor interacts with a different putative scaffolding stood, macroscopic inactivation is not apparent in recombinant protein (Kornau et al., 1995; Niethammer et al., 1996; Brakeman NMDA receptors lacking the intracellular C-terminal domains of et al., 1997; Dong et al., 1997). The subsynaptic scaffold may allow NR1 (Krupp et al., 1998). Several proteins can bind to the the differential anchoring of enzymes close to their site of action intracellular C-terminal domains of NMDA receptor subunits. as occurs in the modulation of AMPA receptor by protein kinase PSD-95, the first such protein to be identified (Kornau et al., A (Rosenmund et al., 1994). Although the evidence provides a 1995; Niethammer et al., 1996), binds via a PDZ recognition motif present at the C terminus of all four NR2 subunits and some NR1 Received Oct. 8, 1998; revised Nov. 10, 1998; accepted Nov. 24, 1998. splice variants. Calmodulin also binds in a calcium-dependent This work was supported by National Institutes of Health Grants MH46613 (G.L.W.) and NS28709 (S.F.H.), the McKnight Foundation (S.F.H.), the John Adler manner to two distinct sites on the C terminus of the NR1 subunit Foundation (S.F.H.), fellowships from the Human Frontiers program (J.J.K., B.V.), (Ehlers et al., 1996). The actin-binding protein a-actinin can bind and the National Health and Medical Research Council of Australia (B.V.). We to the C termini of NR2B and NR1 (Wyszynski et al., 1997), thank the following for cDNA constructs: Drs. A. H. Beggs and M. Sheng (Harvard Medical School; human a-actinin-2), Dr. D. R. Critchley (University of Leicester, providing a potential link between the NMDA receptor and actin Leicester, UK; chicken a-actinins), and Dr. J. P. Adelman (Vollum Institute; CD4). filaments. In vitro, a-actinin and calmodulin bind competitively to The excellent technical support by A. Miller is gratefully acknowledged. Dr. M. Faux kindly helped us with the IAsys optical biosensor system. the initial 30 aa of the C terminus of NR1 (Wyszynski et al., J.J.K. and B.V. contributed equally to this work. 1997). This segment, called C0 (Ehlers et al., 1996), is common to Correspondence should be addressed to Dr. Gary L. Westbrook, Oregon Health all NR1 splice variants (Hollmann et al., 1993). Finally, a novel Sciences University-L474, Vollum Institute, 3181 SW Sam Jackson Park Road, Portland, OR 97201. protein (yotaio) (Lin et al., 1998) and the neurofilament subunit Copyright © 1999 Society for Neuroscience 0270-6474/99/191165-14$05.00/0 NF-L (Ehlers et al., 1998) interact with the alternatively spliced 1166 J. Neurosci., February 15, 1999, 19(4):1165–1178 Krupp et al. • Ca-Dependent Inactivation of Recombinant NMDA Receptors exon cassette C1 of NR1. Except for calmodulin, no data are removed after 10–18 hr by exchanging with fresh culture medium con- available on the physiological function of these interactions. Cal- taining 3 mM kynurenic acid and 1 mMD,L-AP5. FUDR (0.2 mg/ml 59-fluoro-2-deoxyuridine and 0.5 mg/ml uridine, Sigma) was added to modulin reduces the open probability of recombinant NMDA inhibit cell proliferation. receptors in inside-out patches (Ehlers et al., 1996), and it has Before recording, transfected cells were identified by CD4 receptor been proposed that this interaction underlies inactivation (Zhang antibody-coated beads. For bead coating, Dynabeads M-450 CD4 (1 ml, et al., 1998). Dynal, Oslo, Norway) were added to each 35 mm dish and gently swirled We examined the role of the intracellular C terminus of NR1 for 15–20 min before recording. Recording, solutions, and drug application. Whole-cell voltage-clamp and its binding proteins on inactivation of recombinant NMDA recordings were performed 12–48 hr after the end of the transfection. receptors. NR1 constructs were expressed with NR2A in human The recording chamber was continuously superfused at room tempera- embryonic kidney (HEK) 293 cells, and inactivation was moni- ture (;20°C) with an extracellular solution of the following composition tored using whole-cell recording. Our results suggest that the C0 (in mM): NaCl 162, KCl 2.4, HEPES 10, glucose 10, CaCl2 1, pH 7.25 domain directly affects channel open probability in the absence of (NaOH), 325 mOsm. HPLC grade water was used for all solutions to a avoid contaminating amounts of glycine or other amino acids. Patch calmodulin or -actinin binding. We propose that the calcium- pipettes were pulled from thin-walled borosilicate glass (TW150F-6; dependence of inactivation can involve calcium binding to both World Precision Instruments, Sarasota, FL) and had resistances between calmodulin and a-actinin. 2and5MV. The intracellular solution included an ATP-regenerating system (MacDonald et al., 1989; Rosenmund and Westbrook, 1993a) of MATERIALS AND METHODS the following composition (in mM): CsCH4SO3 115.5, HEPES 10, MgCl2 6, Na ATP 4, phosphocreatine 20, creatine phosphokinase 500 U/ml, Molecular biology. The NMDA subunit cDNAs used were NR1–1a 2 leupeptin 0.1, EGTA 0.1, pH 7.2 (CsOH), 320 mOsm (sucrose). In some (accession no. U08261), NR1–1b (accession no. U08263), NR1–2a (ac- experiments EGTA was replaced by 10 mM BAPTA plus 1 mM CaCl as cession no. U08262), NR1–3a (accession no. U08265), NR1–4a (acces- 2 indicated. Patch solutions were prepared daily from frozen stocks and sion no. U08267) (Hollmann et al., 1993), and NR2A (accession no. D13211) (Ishii et al., 1993). NR1 mutants were generated using the kept on ice until use. Drugs and peptides were added to the patch strategy of gene splicing by overlap extension PCR (Horten et al., 1989) solution before the experiment from frozen stock or as powder. Aliquots using Pfu DNA Polymerase (Stratagene, La Jolla, CA). All NMDA of frozen drugs were discarded after single use. If the drug vehicle was subunit cDNAs and chimeras were cloned into pCDNA1/amp (Invitro- DMSO, stock solutions were diluted at least 1:1000. For inside-out experiments, electrodes were filled with Ca-free extracellular solution gen, San Diego, CA). cDNAs generated by PCR were sequenced. NR1 m m truncation mutants were generated by replacing the appropriate codon plus 5 mM EGTA, 5 mM EDTA, 100 M glycine, and 100 M NMDA, pH with a stop codon. The aa numbering in all cases refers to the predicted 7.2 (NaOH).
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