CaMKII binding to GluN2B is critical during memory consolidation

Memory is essential for our normal daily lives and our sense of self. Ca(2+) influx through the NMDA-type glutamate receptor (NMDAR) and the ensuing activation of the Ca(2+) and calmodulin-dependent protein kinase (CaMKII) are required for memory formation and its physiological correlate, long-term potentiation (LTP). The Ca(2+) influx induces CaMKII binding to the NMDAR to strategically recruit CaMKII to synapses that are undergoing potentiation. We generated mice with two point mutations that impair CaMKII binding to the NMDAR GluN2B subunit. Ca(2+)-triggered postsynaptic accumulation is largely abrogated for CaMKII and destabilized for TARPs, which anchor AMPA-type glutamate receptors (AMPAR). LTP is reduced by 50% and phosphorylation of the AMPAR GluA1 subunit by CaMKII, which enhances AMPAR conductance, impaired. The mutant mice learn the Morris water maze (MWM) as well as WT but show deficiency in recall during the period of early memory consolidation. Accordingly, the activity-driven interaction of CaMKII with the NMDAR is important for recall of MWM memory as early as 24 h, but not 1-2 h, after training potentially due to impaired consolidation.     

The subtype of GluN2 C-terminal domain determines the response to excitotoxic insults

It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca(2+) influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD(2B)) and GluN2A (CTD(2A)). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD(2B) enhances NMDAR toxicity, compared to CTD(2A). Furthermore, the vulnerability of forebrain neurons in?vitro and in?vivo to NMDAR-dependent Ca(2+) influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD(2B) exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.     

Brain-derived neurotrophic factor regulates AMPA receptor trafficking to post-synaptic densities via IP3R and TRPC calcium signaling

The change in the number of post-synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamatergic receptors (AMPARs) by neuronal activity is recognized as a molecular basis of synaptic plasticity. Here, we show that Ca(2+) transients evoked by brain-derived neurotrophic factor (BDNF) induce translocation of a subunit of AMPAR, GluR1, but not NMDAR, to the post-synaptic membrane in cultured cortical pyramidal neurons. Among BDNF-induced Ca(2+) transients, that dependent on IP3R was fully required, while store-operated calcium influx through the non-selective cation channel TRPC (transient receptor potential canonical) was partially required for the GluR1 up-regulation, suggesting that spatial and temporal calcium signaling regulate translocation of GluR1 to the polarized membrane domain.     

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.     

Dendritic NMDA receptors activate axonal calcium channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.     

Withdrawal from Cocaine Self-Administration Alters NMDA Receptor-Mediated Ca(2+) Entry in Nucleus Accumbens Dendritic Spines

We previously showed that the time-dependent intensification ("incubation") of cue-induced cocaine seeking after withdrawal from extended-access cocaine self-administration is accompanied by accumulation of Ca(2+)-permeable AMPA receptors (CP-AMPARs) in the rat nucleus accumbens (NAc). These results suggest an enduring change in Ca(2+) signaling in NAc dendritic spines. The purpose of the present study was to determine if Ca(2+) signaling via NMDA receptors (NMDARs) is also altered after incubation. Rats self-administered cocaine or saline for 10 days (6 h/day). After 45-47 days of withdrawal, NMDAR-mediated Ca(2+) entry elicited by glutamate uncaging was monitored in individual NAc dendritic spines. NMDAR currents were simultaneously recorded using whole cell patch clamp recordings. We also measured NMDAR subunit levels in a postsynaptic density (PSD) fraction prepared from the NAc of identically treated rats. NMDAR currents did not differ between groups, but a smaller percentage of spines in the cocaine group responded to glutamate uncaging with NMDAR-mediated Ca(2+) entry. No significant group differences in NMDAR subunit protein levels were found. The decrease in the proportion of spines showing NMDAR-mediated Ca(2+) entry suggests that NAc neurons in the cocaine group contain more spines which lack NMDARs (non-responding spines). The fact that cocaine and saline groups did not differ in NMDAR currents or NMDAR subunit levels suggests that the number of NMDARs on responding spines is not significantly altered by cocaine exposure. These findings are discussed in light of increases in dendritic spine density in the NAc observed after withdrawal from repeated cocaine exposure.     

Calcium activation of ERK mediated by calmodulin kinase I

Elevated intracellular Ca(2+) triggers numerous signaling pathways including protein kinases such as the calmodulin-dependent kinases (CaMKs) and the extracellular signal-regulated kinases (ERKs). In the present study we examined Ca(2+)-dependent "cross-talk" between these two protein kinase families. Using a combination of pharmacological inhibitors and dominant-negative kinases (dnKinase), we identified a requirement for CaMKK acting through CaMKI in the stimulation of ERKs upon depolarization of the neuroblastoma cell line, NG108. Depolarization stimulated prolonged ERK and JNK activation that was blocked by the CaMKK inhibitor, STO-609; this inhibition of ERK activation by STO-609 was rescued by expression of a STO-609-insensitive mutant of CaMKK. However, activation of ERK by epidermal growth factor or carbachol were not suppressed by inhibition of CaMKK, indicating specificity for this "cross-talk." To identify the downstream target of CaMKK that mediated ERK activation upon depolarization, dnKinases were expressed. The dnCaMKI completely suppressed ERK2 activation whereas dnAKT/PKB or nuclear-targeted dnCaMKIV, other substrates for CaMKK, were not inhibitory. ERK activation upon depolarization or transfection with constitutively active (ca) CaMKI was blocked by dnRas. Additionally, depolarization of NG108 cells promoted neurite outgrowth, and this effect was blocked by inhibition of either CaMKK (STO-609) or ERK (UO126). Co-transfection with caCaMKK plus caCaMKI also stimulated neurite outgrowth that was blocked by inhibition of ERK (UO126). These data are the first to suggest that ERK activation and neurite outgrowth in response to depolarization are mediated by CaMKK activation of CaMKI.     

Cross-talk with Ca(2+) influx does not underlie the role of extracellular signal-regulated kinases in cytotoxic T lymphocyte lytic granule exocytosis

One important mechanism cytotoxic T lymphocytes use to kill target cells is exocytosis of lytic granules that contain cytotoxic agents such as perforin and granzyme. Ca(2+) influx and activation of protein kinase C have been known for many years to be key signals for granule exocytosis. Recent work has suggested that activation of extracellular signal-regulated kinases (ERK), members of the mitogen-activated protein kinase (MAP kinase) family, may be a third required signal. We surmised that the involvement of ERK in lytic granule exocytosis could be mediated through cross-talk with Ca(2+) influx, rather than constituting an independent signal. We tested this idea using TALL-104 human leukemic CTLs as a model system and discovered the following. 1) ERK inhibition caused a modest decrease in the amplitude of increases in intracellular Ca(2+) concentration, but this effect cannot account for the profound inhibition of granule exocytosis. 2) Ca(2+) influx can activate ERK in TALL-104 cells, but this effect does not contribute to ERK activation stimulated by solid phase anti-CD3 monoclonal antibodies. We conclude that cross-talk between ERK signaling and Ca(2+) does not mediate the role of ERK in CTL lytic granule exocytosis.     

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.     

Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.