Calcium/calmodulin-dependent protein kinase II is associated with the N -methyl- d -aspartate receptor

Abstract

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca 2؉ inf lux through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca 2؉ ͞calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKII␣ by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca 2؉ inf lux but also close to ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP. Caϩ ͞calmodulin-dependent protein kinase II (CaMKII) mediates a variety of different cellular responses to Ca 2ϩ influx (1, 2). An important source of Ca 2ϩ influx into neurons is the N-methyl-D-aspartate (NMDA)-type glutamate receptor, which is activated by the excitatory neurotransmitter glutamate (2). NMDA-and ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors are clustered at postsynaptic sites opposing presynaptic neurotransmitter release sites (3, 4). Brief trains of presynaptic highfrequency stimulation efficiently activate NMDA receptors (5), resulting in postsynaptic Ca 2ϩ influx and long-term potentiation (LTP). LTP is a long-lasting increase in neurotransmission thought to represent the physiological correlate of learning and memory (5, 6). The induction of NMDA receptor-dependent LTP requires activation of CaMKII in the postsynaptic neuron (6, 7). CaMKII is enriched at postsynaptic densities (8, 9), where it is well placed for activation by Ca 2ϩ influx through NMDA receptors and subsequent phosphorylation of neighboring AMPA receptors, an event contributing to LTP (7, 10). A kinase anchoring proteins, usually called AKAPs, place cyclic AMP-dependent protein kinase (PKA) next to selected substrates such as AMPA receptors (11), and receptors for activated C kinase (RACKs) are important for subcellular localization of different protein kinase C (PKC) isozymes (12). Crucial information about the subcellular targeting of CaMKII is lacking. NMDA receptors would be ideal postsynaptic adapter sites for CaMKII, where it would have full access to Ca 2ϩ influx through these receptors. Cortical NMDA receptors consist of one or two NR1 and two or three NR2A and 2B subunits whose C termini are intracellular (13-16). We show that CaMKII is directly associated with NR1 a...

Increased phosphorylation of the neuronal L-type Ca 2+ channel Ca v 1.2 during aging

“…Film exposures were taken for 0.5, 1, and 2 min, and the resulting immunosignals were quantified by densitometry (28,33). Doubling the exposure time or the amount of sample loaded onto the gel resulted in the expected increase in the signals for anti-CH1923-2932P and the long and short forms of ␣ 1 1.2 as detected with anti-CNC1.…”

“…The antibody anti-CH1923-1932P, which specifically binds to ␣ 1 1.2 when phosphorylated at serine 1928, was raised against a phosphopeptide consisting of residues 1923-1932 (25). Antibodies against NMDA receptor NR1 were from R. Jahn (Max Planck Institute for Biophysical Chemistry, Goettingen, Germany); antibodies against NR2A and NR2B were from R. J. Wenthold (National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda) (32,33); antibodies against PKA C␣ were from C. S. Rubin (Albert Einstein College of Medicine, Bronx, NY) (26); and antibodies against protein phosphatase-1 inhibitors 1 and 2 were from A. C. Nairn (Yale University, New Haven, CT). Anti-PSD-95 (34) and anti-SAP102 correspond, respectively, to JH62092 and JH62514 in ref.…”

“…For quantification of PSD-95 and SAP102, 50 mg of hippocampal tissue was homogenized in 0.5 ml of 1% deoxycholate buffer before ultracentrifugation (32,37). Supernatants were used either directly for immunoblotting (20 l) or after immunoprecipitation (500 l) with a mixture of anti-NR2A and anti-NR2B antibodies (32,33,37). All blots were exposed for increasing amounts of time (e.g., 30, 60, and 120 sec).…”

Regulation of the Neuron-specific Ras GTPase-activating Protein, synGAP, by Ca2+/Calmodulin-dependent Protein Kinase II

“…One set of regulatory proteins associates tightly with the cytosolic portion of the NMDA receptor (2,3). These include Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) (4,5), which is activated by Ca 2ϩ flux through the receptor, and several proteins that are held near the receptor by the scaffold protein PSD-95 (6,7). A complex containing many of these proteins can be isolated from brain homogenates and is called the postsynaptic density (PSD) fraction (8).…”

Propagation of CaMKII translocation waves in heterogeneous spiny dendrites

“…In its inactive state CaMKIIα tends to be located in the cytosol, whereas CaMKIIβ is weakly actin bound (Shen et al 1998). Following Ca 2+ -induced activation, CaMKII accumulates at post-synaptic sites through binding to NMDA receptors (Gardoni 1998;Leonard et al 1999;Bayer et al 2001). If the calcium signal is relatively weak then this binding is rapidly reversible, whereas for stronger stimulation the synaptic accumulation of CaMKII can persist for several minutes due to autophosphorylation.…”