Apo-Calmodulin Binds with its C-terminal Domain to the N-Methyl-d-aspartate Receptor NR1 C0 Region

Abstract

Calmodulin (CaM) is the major Ca2؉ sensor in eukaryotic cells. It consists of four EF-hand Ca 2؉ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca 2؉ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca 2؉ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca 2؉ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca 2؉ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to fulllength NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2 domain. The Ca 2؉ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca 2؉ . Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca 2؉ binds to CaM. Glutamate is the prevailing excitatory neurotransmitter in the mammalian brain. The ionotropic N-methyl-D-aspartate (NMDA)1 -type glutamate receptor is a major source of Ca 2ϩ influx into neurons in the central nervous system. NMDA receptors in the cerebral cortex largely consist of two NR1 and two or three NR2A and 2B subunits, which are homologous to each other (1-4). Each subunit has an extracellular N terminus, four membrane regions (M1-4), and an intracellular C terminus. M1, M3, and M4 are transmembrane regions but M2 loops into and back out of the plasma membrane at its cytoplasmic face. The C-terminal domain of NR1 contains the 30 residue-long membrane-proximal C0 region common to all NR1 isoforms, followed in some NR1 isoforms by the C1 region (37 residues), which can be spliced in or out, and by either the C2 (38 residues) or the C2Ј (22 residues) region, which is downstream of C2 in the genome. The C2 region can be spliced out but, when present, C2Ј will not be translated because C2 carries its own stop codon. In a negative feedback loop calmodulin (CaM) inhibits Ca 2ϩ

Ca2+/CaM Controls Ca2+-Dependent Inactivation of NMDA Receptors by Dimerizing the NR1 C Termini

“…In contrast to a recent report (Akyol et al, 2004), we found that apoCaM did not interact with NR1. Moreover, we demonstrated that ␣-actinin-2 did not interact with the NR1.…”

“…1 B, lane 4, bottom gel). The absence of CaM 1234 copurification contrasts with a recent report, suggesting that the NR1 CT can bind Ca 2ϩ -free CaM (Akyol et al, 2004). Because CaM 1234 is a mutant that may not have properties identical to apoCaM, we directly examined whether wild-type (WT) CaM could interact with NR1 in the absence of Ca 2ϩ .…”

“…Ca 2ϩ /CaM binding to the NR1 CT is proposed to displace ␣-actinin-2, thereby leading to channel inactivation (Wyszynski et al, 1997;Zhang et al, 1998;Krupp et al, 1999;Leonard et al, 2002). Recently it was suggested that Ca 2ϩ -free CaM (apoCaM) is prebound to the NR1 C0 domain via the CaM C-terminal lobe (Akyol et al, 2004). If apoCaM is prebound to C0, however, ␣-actinin-2 binding to NR1 would be precluded and the displacement model inconsistent.…”

Binding of trifluoperazine to apocalmodulin revealed by a combination of small-angle X-ray scattering and nuclear magnetic resonance

“…This was confirmed by crystallographic and nuclear magnetic resonance (NMR) studies on various target Ca 2+ CaM-binding proteins including MLCK, CaMdependent protein kinase (CaMK) and myristoylated alanine-rich Ckinase substrate (MARCKS) (reviewed in Ikura et al, 2002;Yamniuk & Vogel, 2004;Ikura & Ames, 2006). In contrast, although the interactions of apoCaM have been studied with some target proteins such as neurogranin, cyclic nucleotide phosphodiesterase (PDE), small conductance Ca 2+ -activated K + channel (SK channel), voltagegated sodium channel, MLCK and myosin V (Cui et al, 2003;Yuan et al, 1999;Mori et al, 2003;Tsvetkov et al, 1999;Hill et al, 2000;Bayley et al, 2003;Akyol et al, 2004;Tang et al, 2003;Jin et al, 2005;Houdusse et al, 2006), structural studies are very rare (Izumi et al, 2001;Schumacher et al, 2004).…”

The DREAM Protein Negatively Regulates the NMDA Receptor through Interaction with the NR1 Subunit

“…So far, a number of NR1 or NR2 subunit binding partners have been identified in the postsynaptic density. The NR1 binding proteins include calmodulin (CaM) (Ehlers et al, 1996;Akyol et al, 2004), CaMKII (Leonard et al, 2002), ␣-actinin (Wyszynski et al, 1997;Merrill et al, 2007), tubulin (van Rossum et al, 1999), spectrin (Wechsler and Teichberg, 1998), and neurofilament (Ehlers et al, 1998) and Yotiao (Lin et al, 1998). Here, we identify a new binding partner of the NR1 subunit, DREAM.…”