The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is a key determinant of network properties; is involved in many physiological processes such as motor control, sound localization, or sensory adaptation; and is critically dependent on cytosolic [Ca2+]. However, the underlying molecular mechanisms and the identity of the Ca2+ sensor/effector complexes involved are unclear. We now identify a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca2+] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity.

In this article the calcium/calmodulin-dependent protein kinases are reviewed. The primary focus is on the structure and function of this diverse family of enzymes, and the elegant regulation of their activity. Structures are compared in order to highlight the conserved architecture of their catalytic domains with respect to each other as well as protein kinase A, a prototype for kinase structure. In addition to reviewing structure and function in these enzymes, the variety of biological processes for which they play a mediating role are also examined. Finally, how the enzymes become activated in the intracellular setting is considered by exploring the reciprocal interactions that exist between calcium binding to calmodulin when interacting with the CaM-kinases.

The activity of Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) plays an integral role in regulating synaptic development and plasticity. We designed a live-cell-imaging approach to monitor an activity-dependent clustering of green fluorescent protein (GFP)-CaMKII holoenzymes, termed self-association, a process that we hypothesize contributes to the translocation of CaMKII to synaptic and nonsynaptic sites in activated neurons. We show that GFP-CaMKII self-association in human embryonic kidney 293 (HEK293) cells requires a catalytic domain and multimeric structure, requires Ca 2ϩ stimulation and a functional Ca 2ϩ /CaM-binding domain, is regulated by cellular pH and Thr286 autophosphorylation, and has variable rates of dissociation depending on Ca 2ϩ levels. Furthermore, we show that the same rules that govern CaMKII self-association in HEK293 cells apply for extrasynaptic and postsynaptic translocation of GFP-CaMKII in hippocampal neurons. Our data support a novel mechanism for targeting CaMKII to postsynaptic sites after neuronal activation. As such, CaMKII may form a scaffold that, in combination with other synaptic proteins, recruits and localizes additional proteins to the postsynaptic density. We discuss the potential function of CaMKII self-association as a tag of synaptic activity.

Ca2؉ -calmodulin-dependent protein kinase II (CaMkinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (␣, ␤, ␥, and ␦) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K m for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca 2؉ -calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (␥ > ␤ > ␦ > ␣) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (␤ > ␥ > ␦ > ␣). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.1 is a major downstream effector of Ca 2ϩ signaling in eukaryotic cells. A rise in intracellular Ca 2ϩ concentration leads to binding of Ca 2ϩ ions to calmodulin (CaM), which binds to and activates CaM-kinase II. Upon activation, this enzyme has the ability to autophosphorylate, a process that confers Ca 2ϩ -independent activity upon the kinase (1) and greatly increases its affinity for CaM (2). Once activated, CaM-kinase II phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity, synaptic vesicle mobilization, regulation of gene expression, regulation of smooth muscle contractility, and modulation of ion channel function (3-7). The fact that CaM-kinase II has so many potential substrates raises the question of the relationship between its activation and a specific response to a particular Ca 2ϩ signal. Possibly, the regulated expression of the multiple isoforms of CaM-kinase II confers these unique properties.CaM-kinase II is expressed from a family of four closely related genes, ␣, ␤, ␥, and ␦, each of which produces mRNA that can be alternatively spliced, giving rise to at least 30 different proteins (8, 9). The overall organization of each of the four kinase isoforms is similar: an N-terminal catalytic domain is followed by a regulatory domain that contains an autoinhibitory region and a CaM-binding site, and a C-terminal association domain, through which the subunits interact to assemble into holoenzymes (10). Between the CaM-binding domain and the association domain is a region termed the...

Studies of the structural organization of calcium/ calmodulin-dependent protein kinase II␣ (CaM KII␣) and truncated CaM KII␣ by three-dimensional electron microscopy and protein engineering show that the structures consist of 12 subunits that are organized in two stacked hexameric rings with 622 symmetry. The body of CaM KII␣ is gear-shaped, consisting of six slanted flanges, and has six foot-like processes attached by narrow appendages to both ends of the flanges. Truncated CaM KII␣ that lacks functional domains has a structure that is very similar to the body of CaM KII␣. Thus, the functional domains reside in the foot-like processes, and the association domain comprises the gearshaped core. The ribbon diagram of the bilobate structure of CaM KI fits nicely in the envelope of the foot-like component and indicates that the crevice between the two lobes comprising the functional domains is near the middle portion of the foot. The clustering of the functional domains provides a favorable arrangement for the autophosphorylation reaction, and the unusual arrangement of the catalytic domain on extended tethers appears to be significant for the remarkable functional diversity of CaM KII␣ in cellular regulation.Calcium/calmodulin-dependent protein kinase (CaM K) 1 II phosphorylates Ser and Thr residues in numerous proteins. Its relatively high concentration in brain tissue and its lack of specificity indicate that it has an important role as a kinase in many aspects of neuronal function. Its substrates are involved in neurotransmitter synthesis and release; carbohydrate, lipid, and amino acid metabolism; transcriptional, translational, and cytoskeletal regulation; calcium homeostasis; and receptor and channel function (for reviews, see Refs. 1 and 2).At least four distinct genes that encode isozymes of CaM KII are selectively expressed in different tissues (2). Expression of the ␣ and ␤ isoforms is restricted primarily to the nervous system, and the ␣ isoform is found only in neurons. The abundance of ␣ and ␤ isoforms is both anatomically and developmentally regulated in the nervous system (3, 4), and it has been proposed that the subunit composition of holoenzymes may influence the targeting of the enzyme to distinct subcellular sites, such as the postsynaptic density (5-7). For example, recent investigations have shown that the ␤ isoform is preferentially bound to F-actin in dendritic spines and in the cell cortex and that the ␣ isoform is targeted to these locations when it is co-expressed with the ␤ isoform (8).Studies have implicated the ␣ isoform of CaM KII in neuronal cell function. Electrophysiological and behavioral tests in mice carrying a null mutation for the ␣ isoform showed that this enzyme has a major role in control of neuronal excitability (9) and spatial learning (10). Indeed, this isoform is thought to have a key role in the long lasting synaptic enhancement denoted long term potentiation (11-13).CaM KII is dependent on Ca 2ϩ /calmodulin for activation, and its autophosphorylation has an important r...

Development of biologically relevant crowding solutions necessitates improved understanding of how the relative size and density of mobile obstacles affect probe diffusion. Both the crowding density and relative size of each co-solute in a mixture will contribute to the measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses how excluded volume of dextran polymers from 10 to 500 kDa affect microviscosity quantified by measurements of calmodulin labeled with green fluorescent protein as the diffusing probe. Autocorrelation functions were fit using both a multiple-component model with maximum entropy method (MEMFCS) and an anomalous model. Anomalous diffusion was not detected, but fits of the data with the multiple-component model revealed separable modes of diffusion. When the dominant mode of diffusion from the MEMFCS analysis was used, we observed that increased excluded volume slows probe mobility as a simple exponential with crowder concentration. This behavior can be modeled with a single parameter, β, which depends on the dextran size composition. Two additional modes of diffusion were observed using MEMFCS and were interpreted as unique microviscosities. The fast mode corresponded to unhindered free diffusion as in buffer, whereas the slower agreed well with the bulk viscosity. At 10% crowder concentration, one finds a microviscosity approximately three times that of water, which mimics that reported for intracellular viscosity.

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