REvIEwS
CaMKII: a central molecular organizerof synaptic plasticity, learningand memory
Ryohei Yasuda

1✉, Yasunori Hayashi

2✉ and Johannes W. Hell

3✉

Abstract | Calcium–calmodulin (CaM)-dependent protein kinase II (CaMKII) is the most abundant
protein in excitatory synapses and is central to synaptic plasticity, learning and memory. It is
activated by intracellular increases in calcium ion levels and triggers molecular processes
necessary for synaptic plasticity. CaMKII phosphorylates numerous synaptic proteins, thereby
regulating their structure and functions. This leads to molecular events crucial for synaptic
plasticity, such as receptor trafficking, localization and activity; actin cytoskeletal dynamics;
translation; and even transcription through synapse–nucleus shuttling. Several new tools
affording increasingly greater spatiotemporal resolution have revealed the link between CaMKII
activity and downstream signalling processes in dendritic spines during synaptic and behaviouralplasticity. These technologies have provided insights into the function of CaMKII in learningand memory.
Protomers
Individual protein molecules
in a protein complex.

1
Max Planck Florida Institutefor Neuroscience, Jupiter,
FL, USA.

Department ofPharmacology, Kyoto
University Graduate Schoolof Medicine, Kyoto, Japan.
23Department ofPharmacology, Universityof California, Davis, Davis,
CA, USA.

✉e-mail: ryohei.yasuda@
mpfi.org; yhayashi-tky@umin.ac.jp; jwhell@ucdavis.edu
https://doi.org/10.1038/
s41583-022-00624-2

Calcium–calmodulin (CaM)-dependent protein
kinase II (CaMKII) has remarkable properties andfunctions. When activated, CaMKII undergoes autophosphorylation, thus rendering it active independent of
the calcium–CaM complex (hereafter abbreviated as
Ca2+–CaM), beyond the initial stimulation — endowing
the kinase with a ‘molecular memory’1–3. In the hippo­
campus, CaMKII accounts for about 1–2% of total
protein by mass and about 10% of protein in the postsynaptic density (PSD)4–7. The PSD is a protein-dense
meshwork that constitutes the postsynaptic sites of glutamatergic synapses, typically in dendritic spines, thesmall protrusions of dendrites of excitatory neurons.
In addition to its catalytic functions, CaMKII has
essential structural functions, many of which dependon its autophosphorylation and calcium8. Thus, it has
been proposed that CaMKII acts to a large degree asan activity-dependent structural protein2,8,9. CaMKII
has important roles in synaptic plasticity, including in
long-term potentiation (LTP) and spine structural plasticity, and in the formation of many types of memory,providing a key argument for the hypothesis that LTP
is part of physiological underpinning of memory1,2.
CaMKII is also involved in long-term depression(LTD)10 (reviewed elsewhere3). Impaired CaMKII function is associated with intellectual disabilities, developmental delay and seizure activity, further supporting its
crucial roles in synaptic plasticity11–14.
The past decade has seen important progress inunderstanding the mechanisms underlying CaMKIINaTure Reviews | Neuroscience
0123456789();:signalling in synaptic plasticity, in part because newtools have enabled CaMKII signalling to be imaged
and manipulated at the level of single synapses andeven down to the nanometre scale. In addition, recent
structural and biochemical studies have provided fresh
insights into the fine mechanistic details of how CaMKII
works. The goal of this Review is to integrate recent findings that shed new light on postsynaptic CaMKII regulation and function with earlier insights. Important new
tools and concepts include the development of photo­activatable constructs of CaMKII and one of its inhibitors,
the realization of the importance of liquid–liquid phaseseparation (LLPS) for postsynaptic CaMKII functions
and the recognition of a continued binding groove thatis shared by substrates, as well as CaMKII-associatedproteins (CaMKAPs) and some CaMKII inhibitors.

Structure and activity
CaMKII possesses several unique features, including
its ability to be acutely activated by Ca2+–CaM and to
be more enduringly activated by autophosphorylation, as well as its multimeric structure. This section
introduces the structure and mechanisms of activationof CaMKII.
Structure and subunits of CaMKII. CaMKII protomers
consist of a catalytic domain with a bilobed structure(as is typical for kinases), an autoinhibitory or regulatory
segment, a linker of various lengths and an association
domain, which mediates the formation of dodecameric

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a Inactive
CaMKII
subunitb Active
KinasedomainCaM

Regulatory
domainAssociation
domainc Activation scheme
Partially active

Ca2+
T286
P

P

Fig. 1 | structure of caMKii and its activation scheme. a | Structure of calcium–calmodulin (CaM)-dependent protein
kinase II (CaMKII). The holoenzyme consists of 12 subunits, each of which acts as a kinase. Each subunit includes anassociation domain, a regulatory domain and a kinase domain. For simplicity, CaMKII is drawn as a hexamer, but actually
it is a dodecamer consisting of two hexameric rings stacked on top of each other, with both rotational and planar symmetry.
b | Upon activation, each subunit changes its conformation from the closed form to the open form. c | The conformationalchange to CaMKII upon calcium–CaM binding and subsequent phosphorylation of T286.
holoenzymes 15,16 (Fig. 1) . The CaMKIIα, CaMKIIβ,CAMKIIγ and CAMKIIδ subunits are, respectively,
encoded by four different genes — CAMK2A, CAMK2B,
CAMK2G and CAMK2D. The subunits can copolymerize to form either homomeric or heteromeric holoenzymes. CaMKIIα is expressed in excitatory neurons
in the hippocampus and cortex and in some inhibitory
neurons such as Purkinje cells in the cerebellum andmedium spiny neurons in the striatum, whereas othersubunits are expressed more widely among brain regions
and neuronal cell types17. CaMKIIα, CaMKIIβ, and
CaMKIIγ have roles in synaptic plasticity18–21. CaMKIIαand CaMKIIβ are two of the most abundant PSD proteins in the hippocampus, making up approximately
10% of total protein in the PSD (about 960 monomers
per 0.1 μm2 of PSD), which is more than the proto­
typical postsynaptic scaffold protein PSD95 (about
250 monomers per 0.1 μm2 of PSD) in both number
and mass4,7.
Holoenzymes
Fully functional enzymes withall components present.

Activation and autophosphorylation scheme of CaMKII.
Each CaMKII subunit has three critical autophosphorylation sites on its regulatory domain15 — in CaMKIIα,these are at T286, T305 and T306. In the inactive formof CaMKII, access to the catalytic domain is blocked
by the autoinhibitory pseudosubstrate segment of theprotein15,16,22 (Fig. 1). Binding of Ca2+–CaM to the adjacent regulatory segment removes the autoinhibitory
segment from the catalytic site. The simultaneousbinding of Ca2+–CaM to two neighbouring subunits
triggers CaMKII autophosphorylation at T286, whichlies in the autoinhibitory domain23, bestowing kinase
activity that persists after Ca2+–CaM dissociates from
CaMKII (refs.24,25) (Fig. 1). This so-called autonomous
CaMKII activity has a crucial role in translating transient Ca2+ spikes into lasting CaMKII activation26. T286
phosphorylation also increases the affinity of CaMKII
for Ca2+–CaM by between 10-fold and 1,000-fold27,28.
Thus, T286 autophosphorylation of multiple subunits
in a holoenzyme provides a nonlinear translation of
Ca2+ spike frequency and amplitude into kinase activity,
in vitro and in neurons27,29. By contrast, the other autophosphorylation sites, T305 and T306, are located in the
Ca2+–CaM-binding site of the regulatory segment, andtheir phosphorylation prevents Ca2+–CaM binding
and kinase activation15,30,31 (Fig. 1).
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Phosphatases
Enzymes that remove
phosphate moieties from
phosphorylated proteins,
lipids or other substrates.

Bistability
A property whereby asystem can have two stableequilibrium states.

Bulk phase
Bulk phase signalling refers
to signalling mediated bya population of CaMKII thatis generally not anchored to
CaMKAP. This population
senses global calcium ions andtransmits signals to diffusing
substrates, as opposed to local
signaling via CaMKAP–CaMKII
complexes, which occur in
nanodomains.

Glutamate uncaging
A microscopic techniquethat uses photons to release
glutamate from light-sensitive
precursors, often used toinduce long-term potentiationin spines.

CaMKII may exist in distinct populations in dendritic
spines. CaMKII had been thought to provide a mole­cular memory owing to its autophosphorylation at T286
and resultant constitutive activation persisting after
the decline in intracellular Ca2+ concentration26. When theT286 residues of two adjacent subunits are phosphory­
lated, rephosphorylation after phosphatases remove
one of the phosphates can be fast28. This mechanism
potentially causes bistability in the CaMKII autophosphorylation status, and some theories have suggested
that when CaMKII has become autophosphorylated, its
phosphorylation can be maintained for a long time —even years32,33. At the same time, imaging of changes in
the conformation of CaMKII as a proxy for its activity
in dendritic spines has suggested that the activity of
CaMKII, at least in the bulk phase, persists for only abouta minute29,34,35. In addition, most pharmacological inhibitors of CaMKII block LTP when they are introduced
at relatively low concentrations before, but not after,
LTP induction25,36,37. These studies suggest that CaMKII
activity lasts for only a short time compared with thetimescale of memory (Fig. 2).
By contrast, biochemical studies from tissue lysates
suggest that CaMKII is partially active under basal
conditions, and that its activity persistently increases
in response to LTP induction38,39. Some CaMKII inhibitors at high concentrations can reduce basal transmission and impair LTP maintenance, supporting thepresence of active CaMKII under basal conditions40,41.
The CaMKII pools in spines that mediate these effects
might be small and functionally defined by prolonged
interactions of CaMKII with its various binding partners that enable them to contribute to the maintenanceof basal transmission and long-term memory42–44. For
example, CaMKII associates with the NMDA receptor(NMDAR) subunit GluN2B for an extended period,
potentially having structural and/or catalytic roles in
the maintenance of basal transmission and synaptic
memory1–3,42,43,45. Given there might be about 100-foldmore CaMKII molecules in spines than GluN2B, andthe high binding affinity of these molecules for eachother (nanomolar)5,46, the pool of CaMKII bound with
GluN2B is probably not effectively detected by imaging
and may not be readily accessible to low concentrationsof competitive inhibitors40,41,44. In the following sections, we separately discuss bulk-phase CaMKII, whichis readily accessible by imaging and low concentrationsof competitive inhibitors, and the local pools of CaMKII
that interact with other molecules (Fig. 2).

Bulk-phase CaMKII in dendritic spines
The recent development of Förster resonance energy
transfer (FRET)-based CaMKII sensors and two-photon
FRET and fluorescence lifetime imaging microscopy
techniques has enabled the activity of CaMKII in dendritic spines to be directly monitored during LTP andassociated spine growth29,34,35,47 (Fig. 3). By combination ofthese techniques with two-photon glutamate uncaging48,
which enables spatially and temporally coordinated synaptic activation, important details of CaMKII functionsin spines have been revealed.
Biosensors for CaMKII activity. The most commonlyused CaMKII sensor is Camui, an engineered CaMKII
molecule with fluorophores at its amino and carboxy
termini (N and C termini)47. The distance between

Time
0.1 s

Bulk phase

1s

10 s

1 min

10 min1h

Sensivity

Ca2+
Ca2+–CaM binding
T286 phosphorylation
CaMKII activity

Local phase

Actin polymerization
AMPAR insertion

CaMKII–TIAM1 RAKEC
NMDAR Ca2+

• Camui sensor• 5 μM tatCN21• 5 mM AIP2
• 6–50 μM intracellular
paAIP2 (short exposure)

CaMKII–GluN2B
Phase separation• CaMKII–TIAM1 sensor• 20 μM tatCN21• 1 μM myr-CN27• 1 μM myr-AIP2• 6–50 μM intracellular
paAIP2 (long exposure)Fig. 2 | The hypothetical timescale of caMKii signalling in dendritic spines. Transient increases in calcium levels (lasting
10–100 ms)53 in a dendritic spine trigger cascades of biochemical events that lead to calcium–calmodulin (CaM)-dependent
protein kinase II (CaMKII) signalling. CaMKII is activated by binding of the calcium–CaM complex (Ca2+–CaM) (over thecourse of seconds49,54), and its activation is maintained by subsequent phosphorylation of T286. CaMKII activation occurs
in the bulk phase (blue) and local nanodomain phase (red) in different time domains. The timescale of CaMKII activation in
the bulk phase was measured by imaging and found to be transient (between seconds and 1 min (refs.34,35)). The exact
timescale of local-phase CaMKII activity in dendritic spines is still unknown, but the interaction between CaMKII and
CaMKII-associated proteins (CaMKAPs), such as the NMDA receptor (NMDAR) subunit GluN2B and the RHO guanine
nucleotide exchange factor TIAM1, can persist for more than 30 min (refs.44,45). The extracellular concentration of inhibitors
affecting the bulk-phase and local-phase CaMKII-dependent processes are shown in boxes on the right side — with the
exception of the light-inducible intracellular CaMKII inhibitor photoactivatable autocamtide inhibitory peptide 2 (paAIP2),whose intracellular concentration was 6–50 μM (ref.37). AMPAR, AMPA receptor; CN27, 27-amino acid sequence fromthe CaMKII-inhibitory protein CaMKIIN1; myr, myristoylated; RAKEC, reciprocally activating kinase–effector complex;
tatCN21, tat peptide fused with a 21-amino acid sequence from CaMKIIN1.
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The time when something
decaying in exponential fashion
becomes 1/e, where e is
Napier’s constant.

the fluorophores changes when the CaMKII part of thesensor is activated and undergoes conformationalchanges, enabling FRET-based imaging of its activation (Fig. 1). In vitro assays suggest that this sensor
reports Ca2+–CaM binding as well as T286 autophosphorylation, with the FRET signal upon T286 autophosphorylation being about 60% of that seen with
Ca2+–CaM34,47,49. This difference is qualitatively consistent with biochemical assays that indicate that
CaMKII is several times more active when Ca2+–CaM
is present than when it is T286-phosphorylated but
Ca2+–CaM is removed50,51. One limitation of Camui isthat it measures the conformational change of overexpressed CaMKII as a proxy for CaMKII activity.
A new sensor called ‘FRESCA’ (FRET-based sensor
for CaMKII activity) measures the balance of kinaseand phosphatase activity using a synthetic CaMKII
substrate peptide attached to fluorescent proteins52.
Use of FRESCA in the future should help to define thespatiotemporal dynamics of different CaMKII pools.
Baseline signalling. The percentage of CaMKII in theactive or phosphorylated state in spines has not beenmeasured. However, excitatory neurons exhibit low
resting calcium ion concentrations (20–50 nM), andthus resting CaMKII activity is probably low53,54. Indeed,
Camui-imaging studies show that wild type Camui signal

under resting conditions is similar to that of the T286A
mutant Camui (which cannot undergo T286 autophosphorylation), and the T305D–T306D mutant (whichcannot bind to Ca2+–CaM)49. Thus, under basal conditions, the effects of T286 phosphorylation and CaM binding on CaMKII activity seem to be below the detection
limit of this method (about 10% of CaMKII acti­vation
during LTP)34,35. Sensors with higher signal-to-noise
ratios should provide improved quantitative assessmentsof CaMKII activity under basal conditions.
Calcium level elevation and CaM binding. After calcium ions flow into a dendritic spine, they are extruded
from the spine in 10–100 ms (ref.53). During this period,
CaM binds to Ca2+, and the resultant Ca2+–CaM binds to
CaMKII (ref.54). The association of CaM with CaMKII
was imaged with a FRET-based sensor that consisted ofenhanced green fluorescent protein (eGFP)–CaMKII and
mCherry–CaM. The decay time constant without autophosphorylation was determined to be approximately
1 s (ref.49), consistent with computer simulations54 (Figs. 2
and 3), whereas T286 autophosphorylation prolonged
this decay time to several seconds to minutes (see later).
Autophosphorylation of T286. When two adjacent
CaMKII subunits bind Ca2+–CaM, they undergo autophosphorylation at T2861,23,35,55,56 (Fig. 1), prolonging theiraGlutamate uncaging (0.49 Hz, 30 pulses)
1st
2nd

–0.4

3rd

4th

0.1

0.6

1.2

1.7

2.2

2.7

3.2

3.7

4.2

4.7

5.2

5.8

6.3

6.8

1 μM
7.3

60.0

60.5

61.0

61.5

62.0

62.5

63.0

63.6

64.1

64.6

65.1

65.6

66.1

66.6

67.1

30th

59.5

Time (s)1.6

2.1

Fluorescence
lifetime (ns)bGlutamate uncaging

sLTP induction

Change in CaMKII activity

Decay time constant

10 s

CaMKII activity
CaMKII–CaM association
Calcium levels

10 sFig. 3 | Activation of caMKii in single dendritic spines. a | Activation of calcium–calmodulin (CaM)-dependent kinase II(CaMKII) imaged by two-photon fluorescence lifetime imaging microscopy and the Camui CaMKII sensor. CaMKII activity
in single dendritic spines can be imaged with subsecond temporal resolution using the Camui sensor during spine structural
long-term potentiation (LTP). b | CaMKII activation time course compared with CaM–CaMKII interaction and increases incalcium levels. CaMKII activity accumulates over the course of about 10 s, whereas the CaM–CaMKII interaction is saturated
in about 1 s (ref.49). This difference is due to the autonomous, phosphorylation-dependent but Ca2+–CaM-independent
CaMKII activity. Part a is adapted with permission from ref.35, Elsevier. Part b is adapted with permission from ref.49, Springer
Nature Limited.
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A-type CaMKAPs

Actinﬁlament

F-type CaMKAP

Active CaMKII

Ca2+Inactive
CaMKII

Fig. 4 | Activity-dependent caMKii association changes the localization of caMKAPs during long-term potentiation.
Calcium–calmodulin (CaM)-dependent kinase II (CaMKII, shown in teal) is associated with various proteins in an activitydependent manner. Increases in the level of calcium in spines can trigger changes in the localization of CaMKII and
CaMKII-associated proteins (CaMKAPs) during synaptic plasticity. Anchoring-type (A-type) CaMKAPs, including F-actin(actin filaments shown in dark grey) and NMDA receptor subunit 2B (GluN2B), define the localization of CaMKII, whereas
follower-type (F-type) CaMKAPs, including the RAC guanine nucleotide exchange factor TIAM1, activity-regulated
cytoskeleton-associated protein (ARC) and proteins of the proteasome, are recruited by CaMKII.

Structural LTP(sLTP). An activity-induced,
lasting increase in spine size,
mirroring electrically induced
long-term potentiation (LTP).

binding to Ca2+–CaM severalfold in spines49, consistent
with cell-free assays27. In addition, autophosphorylation at T286 enables CaMKII to integrate calcium signalling over about 6 s (refs.35,49) (Figs. 2 and 3). Given
that there is no increase in CaMKII–CaM association
beyond about 1 s after the decline in Ca2+ concentration,
most of this integration over seconds is perhaps due tothe autonomous activity of CaMKII, as overall CaMKII
activation continues to increase over seconds to minutes during CaMKII–CaM association35,49 (Figs. 2 and 3).
CaMKII deactivates with a time constant of about 1 min,which is slower than that for Ca2+–CaM association (1 s)and the fast component of CaMKII activity decay (about
6 s)35,49. The biochemical mechanism of this slow decay
is not clear, but it could be explained by an additional
phosphorylation state or association with other proteins that protect CaMKII from phosphatase action49.
Inhibitory phosphorylation at T305 and/or T306 seems
to have only minor effects on the rate of decay49.
T286 phosphorylation seems to prolong CaMKII
activation by severalfold, enabling CaMKII to integrate
short periods of calcium signalling (~100 ms). In otherwords, T286 phosphorylation regulates the time constantof the integrator function of CaMKII (Fig. 3). Because thedecay time constant of T286A-mutant CaMKIIα is about
1 s, it is possible to induce LTP or spine enlargement in
CaMKII T286A knock-in mice by stimulating spines athigher frequency (for example, with glutamate uncaging at 8 Hz in low extracellular Mg2+ conditions, or with
40-Hz electrical stimulation of depolarized postsynaptic neurons)35. Structural plasticity of dendritic spines
can also be induced in CaMKII T286A knock-in miceby high-frequency stimulation, even when CaMKIIβexpression is also knocked down35. These findings suggest that CaMKII T286 phosphorylation is required tointegrate the activity of CaMKII beyond the duration of
Ca2+ concentration elevations but not to maintain LTP
and structural plasticity.
Timescale of endogenous CaMKII signalling in dendritic
spines. Imaging CaMKII activity provides the timescaleof CaMKII activity in single dendritic spines. However,

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0123456789();:the timescales of the activity of overexpressed exogenous
sensors may differ from those of endogenous CaMKII
activity. Compounds that inhibit substrate phosphorylation by CaMKII generally inhibit LTP induction but
do not reverse established LTP25,36, suggesting that thetimescale of endogenous CaMKII activity in spines isroughly consistent with imaging results34,35. The recent
development of the genetically encoded photoinducible
CaMKII inhibitor photoactivatable autocamtide inhibitory peptide 2 (paAIP2) has enabled the timescaleof CaMKII signalling to be resolved37. Photoactivation of
paAIP2 inhibits LTP and structural LTP (sLTP)48 during
their induction, but not when the photoinhibition of
CaMKII is delayed by 1 min, suggesting that CaMKII
activation is required for only about 1 min during
induction37, consistent with imaging results34,35. Thus,
two methods — imaging and photoinhibition — havedetermined the timescale of CaMKII in the bulk phase
to be in the range of seconds to about 1 min.

Local population action of CaMKII
Kinases are slow enzymes (typically carrying out 0.1–10
reactions per second); thus, tethering kinases near theirsubstrates is a key strategy for achieving the necessary
efficiency of the kinase action57,58. Carrying out 10
reactions per second50, CaMKII is no exception. Many
CaMKAPs bind to the substrate-binding region of
CaMKII, affecting synaptic plasticity (Fig. 4). The highabundance of CaMKII (about 100 µM) at the postsynaptic site enables even a relatively weak interaction to cause
a substantial degree of association. Given its dodecameric
structure, CaMKII can crosslink various postsynaptic
CaMKAPs, including GluN2B, densin 180 (also known
as leucine-rich repeat-containing protein 7 (LRRC7)) and
α-actinin59. Some interactions are induced by Ca2+–CaM,
whereas others (such as those with F-actin) are disrupted
by it. Notably, for some proteins, including GluN2B and
TIAM1, the Ca2+–CaM-induced interaction can persist
even after dissociation of Ca2+–CaM42,44,60. Hence, simi­
larly to T286 phosphorylation, such protein–protein
interactions have been proposed to provide anothermechanism for molecular memory during LTP61.

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Guanine nucleotide
exchange factor(GEF). A protein that activatessmall GTPases such as RAS and
RHO by exchanging GDP on
the small GTPases with GTP.

Condensates
Collection of molecules
separated and condensed fromthe dilute phase through theprocess of liquid–liquid phaseseparation.

Apo state
The state in which an enzyme
lacks one or more constituentsrequired for its activity.

Ultrastructural distribution
Distribution of molecules
at the nanometre scale,as determined by electron
microscopy.

Exchange time
The time required formolecules in a compartment
to be replaced with molecules
in another compartment.

CaMKAPs can anchor CaMKII at specific nanodomains, such as the PSD or cytoskeleton, or can be follower or client proteins that are recruited to these sites
through CaMKII binding. In this Review, we use theterm ‘anchoring (A)-type CaMKAPs’ for the formerand ‘follower (F)-type CaMKAPs’ for the latter. A-type
CaMKAPs include GluN2B42, L-, T- and P/Q-type
voltage-gated calcium channels62–65, F-actin66–68 andmicrotubule proteins69. F-type CaMKAPs include the
RAC guanine nucleotide exchange factor (GEF) TIAM1(ref.44), activity-regulated cytoskeleton-associated protein(ARC)70 and proteasomal proteins71,72, and are important for producing local nanodomain signalling20,34,63.
Densin 180 and α-actinin can act as F-type or A-type
CaMKAPs2,73 (Fig. 4).
A unique CaMKAP is the L-type calcium channel
CaV1.2. CaMKIIβ interacts with Cav1.2 and locallyphosphorylates CaMKIIγ, facilitating the binding of
Ca2+–CaM to CaMKIIγ. Upon arrival of the complex
at the nucleus, Ca2+–CaM is released from the complex. Thus, through these reactions, CaMKIIγ shuttles
Ca2+–CaM into the nucleus, where Ca2+–CaM stimulates cAMP response element-binding protein (CREB)
phosphorylation via CaMK kinase (CaMKK) and CaMK
type IV (CaMKIV)20.

Remarkably, when CaMKII undergoes LLPS with
AMPARs and NMDARs, the overall status of the droplet changes dramatically upon binding of Ca2+–CaM to
CaMKII, separating NMDAR-rich and AMPAR-rich
subdomains within the protein condensates that form
during LLPS (Box 2). Furthermore, CaMKII can undergo
LLPS with the synaptic protein SHANK3 (SH3 andmultiple ankyrin repeat domains protein 3) without
a need for Ca2+–CaM or any of the aforementioned
proteins79. Therefore, CaMKII can form several different condensates in spines depending on its activation
status.
Interestingly, in its inactive apo state CaMKII binds
to the N-terminal region of SHANK3 (ref.79), but when
activated by Ca2+–CaM, it binds instead to a central
polybasic motif (Arg949-Arg950-Lys951)80. One possible interpretation of these data is that calcium-ion influx
can induce a conformational change of the pre-existing
CaMKII–SHANK3 complex, driving additional protein
interactions and restructuring of the nanoscale environ­ment of SHANK3. Phosphorylation of SHANK3 by
CaMKII may play a part here81. These changes could
occur at the sites of pre-existing CaMKII–SHANK3
complexes or could induce a redistribution of CaMKII
from the spine interior to the PSD82–84.

CaMKAPs and LLPS of CaMKII. The excess of CaMKII
compared with GluN2B leaves a large number of kinasesubunits that are available for binding with otherproteins. Indeed, the number of identified CaMKAPs(A-type and F-type) is increasing, and currently includes
TIAM1, densin 180, the L-type Ca2+ channel CaV1.2
and the small GTPase REM2 (refs.42,44,73–75). Comparisonof the amino acid sequence in these CaMKAPs that bind
CaMKII in recent crystallographic analysis46,76,77 reveals
a consensus sequence of (R/K)-Xn-L-X-(R/K)-Q-X(S/T)-φ-D (where n is 2 or 3, and φ symbolizes a hydrophobic amino acid; Box 1). Notably, this overlaps withconsensus phosphorylation site RXX(S/T), although not
all of the binding partners of CaMKII are necessarily
substrates, as some feature alanine instead of serine/
threonine. Because there are more than 400 known
CaMKII consensus phosphorylation sites (Box 1 Figure
part c), it is likely that there are more CaMKAPs thatremain to be identified.
LLPS is emerging as a driving force for the formation of localized nanodomains that include CaMKII
as well as AMPA receptors (AMPARs) and NMDARs.
Weak and multimeric interactions between CaMKII andvarious CaMKAPs can produce condensates78,79. Also,
CaMKII can change its binding partner depending on
the activation status79. We postulate that the LLPS of
CaMKII has two key roles in synaptic plasticity. First,
through interactions with A-type CaMKAPs through thesubstrate-binding site, CaMKII is anchored and forms
a condensate beneath the postsynaptic membrane in a
calcium-dependent manner, trapping F-type CaMKAPs
in the condensate through interactions via the same
substrate-binding site. In this way, CaMKII may serve
as a ‘sponge’ to sequester various postsynaptic proteins,thereby triggering Ca2+-dependent trafficking of various
proteins61.

Activity-dependent localization changes of CaMKII.
Increasing calcium levels globally — through bathapplication of glutamate, NMDA or KCl — induces
activity-dependent binding of endogenous CaMKII to
NMDARs42,85 and redistribution of CaMKII from thedendritic shaft to spines86,87. This redistribution depends
on the activity-driven binding of CaMKII to GluN2B42,88
but little on T286 phosphorylation42,89. At the nanometre
scale, redistribution also seems to occur from the spineinterior to the PSD. Electron micrography has shown
that, under basal conditions, the CaMKII distribution
peaks about 40 nm from the PSD. By contrast, spines
stimulated with high-concentration potassium solution
display more CaMKII near the PSD, whereas those inactivated by chelating extracellular calcium exhibit more
cytosolic CaMKII distribution83,90.
A super-resolution microscopy technique called‘single-particle tracking photoactivated localization
microscopy’ enables the ultrastructural distribution of
CaMKII in dissociated live neurons to be measured
without fixation artefacts. A study using this technique91
found that CaMKII is distributed both at the PSD
and away from the PSD, and that NMDAR activation
increases the pool of CaMKII at the PSD, consistent withelectron microscopy studies83. Furthermore, mobility
analyses suggest that CaMKII is immobilized in spines
both at and away from the PSD, probably throughinteraction with PSD-resident proteins and F-actin,
respectively92.
The diffusion constants measured by single-particle
tracking broadly range from 10–3 to 10–1 μm2 per secondand define multiple populations of CaMKIIα (ref.91).
The fast mobile pool (about 10–1 μm2 per second) is
consistent with the exchange time of the highly mobile
pool of CaMKII as measured by fluorescence recovery
after photobleaching (about 1 min)34,62,93,94. This poolwww.nature.com/nrn

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Reviews
Box 1 | structural basis of activity-dependent binding of caMKii and binding partners
Early studies proposed that calcium–calmodulin (CaM)-dependent kinase II(CaMKII) has two discrete binding sites: the substrate-binding site (S-site)
and the T-site, which is where the region of the autoinhibitory domainaround T286 docks42,89. Some CaMKII-associated proteins (CaMKAPs),
including GluN2B, can bind both to the T-site and to the S-site. After
GluN2B initially binds to the S-site and is phosphorylated, it dissociatesfrom the S-site and binds to the T-site89. This model explained thebiochemical assays that have suggested that GluN2B binding to the T-site
can lock CaMKII into an active conformation89, with the S-site remaining
open for substrate binding. However, recent studies resolving X-ray
crystallographic structures of the CaMKII kinase domain bound to GluN2B
and other peptides may force us to consider an alternative model.
The resolved structures include those of CaMKIINtide, which is derived
from the CaMKII-inhibitory protein CaMKIIN1 (Protein Data Bank (PDB)
identifier 3KL8)77, the Drosophila potassium voltage-gated channel protein
dEag (PDB identifier 5H9B)76, GluN2B (which binds to the T-site and the
S-site of CaMKII; PDB identifier 7UJP), TIAM1 (which binds to the T-site of
CaMKII; PDB identifier 7UIR), densin 180 (also known as leucine-richrepeat-containing protein 7 (LRRC7), which binds to the S-site of CaMKII;PDB identifier 6X5G) and GluA1 (which contains a S831 phosphorylation
site; PDB identifier 6X5Q)46. An alignment of these structures showsthat they all bind CaMKII essentially in the same manner (see the figure,a

GluN2B
TIAM1
LRRC7

parts a,b, and ref.46). When the amino acid sequence of CaMKII-binding
proteins is aligned to 0 at a phosphorylation site in CaMKII, the criticalresidues that these proteins tend to share are R/K at –8, L at –5, R at –3, Q at
–2 and φ at +1. Upon alignment of known CaMKII phosphorylation sites of
its substrates from the database PhosphoSitePlus, the consensus sequenceof L-X-R-Q/R-X-S/T-φ-D emerges (see the figure, part c), consistent withkey residues of T-site-binding peptide from crystallographic data.
The crystallographic data show that there is one continuous binding site,rather than discrete S-sites and T-sites, which also explains why ATP binding to the catalytic S-site enhances binding to GluN2B160. It remains unresolved how GluN2B and TIAM1 lock CaMKII into an active conformation.
The crystal structures46,76,77 also do not explain why binding some proteins,
such as CaMKIIN1 and LRRC7, inhibits the kinase activity of CaMKII,
whereas GluN2B and TIAM1 binding does not. Because these crystallographic data were obtained with a truncated monomeric form of CaMKII(residues 7–274), it is conceivable that the binding of some CaMKII sub­units to certain (but not all) CaMKAPs induces an overall conformationof the CaMKII holoenzyme that enables CaMKII subunits that are notengaged in CaMKAP binding to become active independently of calcium–
CaM binding or T286 phosphorylation. AIP2, autocamtide inhibitory
peptide 2; C lobe, carboxy-terminal lobe; N lobe, amino-terminal lobe.
Figure adapted with permission from ref.46, Elsevier.
GluA1
CaMKIIN1

N lobe

–
φ

φ φ

φ

+
L

–
+
–8

φ

φ

–3

–5

φ
φ

ATP
C

φ

b

Phosphorylation
–3
–8
–5
–2
+1
GluN2B – – – – – – – – R – N K – L – – R – R Q – H S Y D T
TIAM1 K T L D S H A S R M A Q – L – – K – K Q – A A L S G
LRRC7 – – – – – S H G R – R P – L – – I – R Q D R I V G V
CaMKIIN1 – – – – – – – K R – P P K L G Q I G R S – K R V V I
dEag – – – – – – – – Q – A T – L – – A – R Q – D T I D E
GluA1 – – – – S K R M K – G F C L – – I – P Q – Q S I N E
CaMKIIα – – – – S H R S T V A S C M – – H – R Q – E T V D C
AIP2 – – – – – – – – K – – K K L – – R – R Q – E A F D A L

c

R –3

1

φ +1

L –5

+1

Q –2

0

C lobeN

R/K –8418 input sequences–1 –7 –6 –5 –4 –3 –2 –1 0NHydrodynamic radius
Radius of a molecule or proteinwith its full hydration shell,
formed by water molecules.

may represent cytosolic, unbound CaMKII molecules.
However, overall the diffusion speed of CaMKII isslower than that expected from the hydrodynamic radius(about 10 μm2 per second)95,96. The low-mobility fractionof CaMKII probably includes the populations boundto immobilized F-actin or PSD proteins as a resultof LLPS67,91,92,97–99.
Overall, these nanometre-scale studies suggest that
CaMKII has at least three modes of distribution — one
cytosolic, one at the PSD and the other near F-actin —and that the balance in distribution of the CaMKII
pool between these loci shifts towards the PSD in an
NMDAR-dependent manner 86. Further molecularstudies have suggested that CaMKII localization at the
PSD is mediated at least to a substantial degree by
the interaction between GluN2B and CaMKII (refs.88,89),
whereas CaMKII localization at the cytoskeleton ismediated through a CaMKIIβ–F-actin interaction66,92,100.

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1

2

3

4

5

6

7

C

In the following sections, we discuss specific interactions
in detail.
Cytoskeleton. The CaMKIIβ subunit interacts with theactin cytoskeleton through its unique linker sequenceand probably through several other sites67,97–99. Because atypical neuronal CaMKII holoenzyme can include
multi­ple CaMKIIβ subunits, this interaction bundles andstabilizes actin filaments in spines66,67 and probably contributes to the localization of CaMKII inside spines19,101.
Ca2+–CaM binding to the regulatory domain of CaMKII
as well as the autophosphorylation at multiple serines or
threonines in the F-actin-binding region of CaMKIIβreduces the affinity for F-actin92. Therefore, CaMKIIβ isreleased from F-actin upon calcium-ion influx intospines, thereby creating a time window in which F-actin
can be modified by actin-regulatory proteins66,92,100. Thus,
CaMKIIβ activation gate sLTP and CaMKII–F-actin

Reviews
PDZ domain
A protein domain that includes
a shallow groove that binds the
carboxy termini of definedproteins that typically end with
Ser/Thr-X-Val, with X being anyof the 20 protein-forming
amino acids.

binding could provide a pool of CaMKII inside spinesthat can relocate moderately quickly during early phasesof LTP. Consistent with this, mice expressing a variant of
CaMKII that is constitutively phosphorylated at theseF-actin-binding sites show impairments in structural
plasticity and learning60. In addition, CaMKII can interact
with microtubules in an activity-dependent but transientmanner69. The interaction of CaMKIIα with α-actinin
may also contribute to its interaction with F-actin2,73.
GluN2B subunit of NMDARs. The interaction between
GluN2B and CaMKII is important for LTP2,40,43,88,102. This
interaction requires CaMKII to be in an open conformation, induced by either Ca2+–CaM binding or T286
phosphorylation42. Thus, this interaction is driven by
Ca2+ influx and consequent Ca2+–CaM binding and
CaMKII activation. During LTP, it mediates recruitment of CaMKII to the PSD, where NMDARs reside88.

Hippocampal LTP is attenuated in knock-in miceexpressing a variant of the NMDAR subunit GluN2B
with crucial interaction sites mutated (L1298A andR1300Q) 88 or when mutant GluN2B (R1300Q and
S1303D) is overexpressed in organotypic slices102. Upon
calcium influx, as occurring during LTP, GluN2B binding strategically localizes CaMKII at the source of calcium influx and near AMPARs, the ultimate effectors
in synaptic transmission and LTP. Indeed, in GluN2B
L1298A–R1300Q knock-in mice, the activity-driven
phosphorylation of the AMPAR subunit GluA1 on S831
by CaMKII is abolished88.
As stated earlier, CaMKII outnumbers GluN2B
by a large margin in PSDs. How, then, can binding of
CaMKII to GluN2B be so central to recruitmentof CaMKII to the PSD and to LTP? Possible explanations
are that GluN2B-bound CaMKII can recruit additional
CaMKII holoenzymes to the PDZ domain through directBox 2 | Phase separation and caMKii

a
CaMKII
GluN2Bc
DIC
Liquid–liquid phase separation (LLPS) is an emerging concept
in cell biology. Solutions containing biological macromole10 μm
cules, such as proteins and nucleic acids, spontaneously sepa–
184,185rate into condensed and diluted phases. The condensation
CaMKIIof a select set of molecules in a small volume leads to the
DIC
T286A
GluN2Bc
formation of membraneless compartments in the cell. Thosemole­cules retain their native conformation and activity, andCa2+
undergo dynamic exchange between diluted and condensed
phases. The condensate retains properties of a liquid, distinct
from protein aggregates, which are typically solid and can be
irreversible.
Ca2+ →LLPS often occurs among molecules with weak
EGTA
multivalent interactions184,185. Indeed, calcium–calmodulin(CaM)-dependent kinase II (CaMKII) simultaneously interacts
with multiple CaMKII-associated proteins (CaMKAPs) throughb
DIC
CaMKII
STGc
GluN2Bc
PSD95
its dodecameric structure. One study tested whether CaMKII
undergoes LLPS with the carboxy-terminal tail of one of its
associated CaMKAPs, the NMDA receptor (NMDAR) subunit–
GluN2B (GluN2Bc)78. The mixture of CaMKII, GluN2B andcalcium-free CaM did not undergo LLPS (see the figure, part a);however, when calcium was added, the proteins formed
condensates within 30 s. Once formed, the condensate
Ca2+
persisted — even after calcium was chelated with use of
EGTA — as long as ATP was present during the initial
incubation to enable T286 autophosphorylation. However,whether CaMKII indeed undergoes LLPS at the synapse
Ca2+ →
EGTA
remains to be determined.
The carboxy-terminal tail of TARPγ2, also known as stargazin(STG), serves as a proxy for the AMPA receptor (AMPAR)
synapse as well. In this way, LLPS of CaMKII serves as a mechanism tocomplex. When this carboxy-terminal tail (STGc) and the scaffolding
translocate to defined nanodomains in the synapse not only CaMKII
protein postsynaptic density protein 95 (PSD95) were added to theitself but also other proteins that bind CaMKII, such as densin 180 and
mixture78, STGc, GluN2B and PSD95 underwent LLPS, but CaMKII
remained in the diluted phase in the absence of calcium. When calcium
α-actinin186 (Fig. 4). Notably, in this context, the CaMKAP α-actinin hasa central role in anchoring PSD95 and thereby AMPARs in spines187 in a
was also added to the mixture, CaMKII accumulated in the condensate.
dynamic manner. Accordingly, NMDAR-mediated calcium-ion influx
Intriguingly, STGc and PSD95 segregated into another phase inside
the CaMKII–GluN2B condensate (see the figure, part b).
induces binding of the calcium–CaM complex to PSD95, displacing
PSD95 from α-actinin188. This displacement causes PSD95 to diffuse
Super-resolution microscopy has revealed that AMPARs and NMDARs
out of spines188. Because PSD95 anchors AMPARs at postsynaptic sites,
exist in distinct nanodomains of the synapse132,133. Blockade of CaMKII–
GluN2B disrupted LLPS and reduced segregation of AMPARs andthis loss of PSD95 leads to a loss of AMPARs from spines, as observed
NMDARs78. Accordingly, LLPS of CaMKII might mediate not only theduring homeostatic synaptic downscaling188 and long-term depression189.
accumulation of CaMKII beneath the synaptic contact but also
That α-actinin also binds CaMKII might be important for the CaMKIIthe formation of glutamate receptor nanodomains at the surface.
mediated phosphorylation of S73 of PSD95, which contributes to the
The dodecameric structure of CaMKII, and its abundance, whichdisplacement of PSD95 from spines upon prolonged Ca2+ influx190.
exceeds that of GluN2B at the synapse, enables other proteins to join
Scale bar, 10 µm. DIC, differential interference contrast. Figure adapted
the condensate as follower proteins, thereby enriching them at thefrom ref.78, Springer Nature Limited.
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Reviews
Antiparallel coiled coil
A protein structure formed by
two α-helices facing each otherside by side in an antiparallelway; for example, by one helix
oriented amino terminus tocarboxy terminus and theadjacent helix oriented
carboxy terminus to amino
terminus.

Hebbian LTPLong-term potentiation (LTP)
that occurs when postsynaptic
activity is paired with
presynaptic activity.

Virus-like capsid
A protein structure that
resembles the capsid structurethat surrounds the viralgenome.

binding, through LLPS or by signalling beyond the
NMDAR complex.
When a CaMKII subunit is bound to the C-terminal
tail of GluN2B, the binding protects CaMKII from reclosure, maintaining the constitutive activity CaMKII onother substrates42. Because this binding facilitates the
CaMKII catalytic activation, rather than competing,
the GluN2B–CaMKII interaction has been hypothesized to occur in a binding pocket different from thesubstrate-binding pocket42,89. This site has been namedthe ‘T-site’ (after the basal-state T286-docking site)
as opposed to the substrate-binding site (‘S-site’)42,89.
However, work showing the crystal structures of the
CaMKII kinase domain bound to different effector proteins suggests that the core CaMKII-binding sequenceof the GluN2B C-terminal tail binds to a rather extensive continuous region on CaMKII that includes both
S-site and T-site residues simultaneously46 (Box 1). The
same binding scheme is shared by other substrates andinteracting proteins46,76,77. Thus, multiple CaMKAPs
and substrates compete for the same binding sites. From
the observed crystal structure46, it is unlikely that the
T-site and the S-site are completely distinct.
It is also not clear how GluN2B–CaMKII interaction facilitates catalytic activity of CaMKII, because thebinding occupies the catalytic core. It is possible that
CaMKII binding to GluN2B can facilitate the catalytic
activity of the neighbouring subunit in the holoenzyme.
The autoinhibitory regulatory region of CaMKII(residues 273–317) forms a helix, and the helices of two
adjacent subunits can form an antiparallel coiled coil22,
which masks substrate-binding pockets. GluN2B binding to one of the substrate-binding pockets dissociates
the coiled coil, possibly affecting the stability of thestructure. This might impair the inhibitory activity ofthe other helix in the dimer in trans, and thus facilitate substrate binding. However, this model does not
explain why GluN2B and TIAM1 can promote andmaintain activity of CaMKII, whereas other substrates inhibit the kinase activity of CaMKII. In addition, electron microscopy has shown that most of thekinase domains are monomeric and less than 20% aredimeric16. Obviously, further refinements and validation
are needed to address how CaMKII–GluN2B binding
promotes CaMKII activity.
Proteasome. The proteasome is a large protein complex
that mediates protein degradation. It accumulates instimulated spines during LTP and is thought to remove
regulatory proteins as well as structural proteins thatwould otherwise hamper the remodelling of spines,
to enable spine growth71. The proteasome binds to
CaMKII, and its activity-driven postsynaptic accumulation requires CaMKII binding to GluN2B71,72. One protein that is degraded by the GluN2B–CaMKII-anchoredproteasome is ephexin 5, which is a RHOA GEF thatcounteracts spine formation103. Other potential targetsof the GluN2B–CaMKII-anchored proteasome include
ARC104,105, the GTPase-activating proteins RAP1 and
RAP2 and their upstream activator RAPGEF4 (alsoknown as EPAC2)106,107, the cyclin-dependent kinase
CDK5 and the Polo-like kinase PLK2 (ref.108). These

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Reciprocal kinase–effector complex with TIAM1.
TIAM1, which promotes F-actin elongation by acting
as a GEF and thereby an activator for the small G protein RAC, interacts with CaMKII in a manner similar
to GluN2B during LTP44. It associates with CaMKII ina Ca2+–CaM-dependent manner, and this binding locks
CaMKII in an open, active state for an extended timeeven after calcium is removed, similarly to GluN2B. In
this way, TIAM1 can activate CaMKII independently of
T286 phosphorylation.
During binding, TIAM1 is phosphorylated at multi­
ple sites by CaMKII. This phosphorylation activates
the GEF activity of TIAM1 (ref.109). Thus, these twomolecules potentially generate a reciprocally activating
loop through binding as long as the complex persists.
This mechanism, termed ‘reciprocal activation within akinase–effector complex’, can convert a transient calcium
signal into persistent activation of both CaMKII and thedownstream molecule. A FRET-sensor study measuring
the dynamics of the reciprocally activating kinase–effector complex44 revealed that this complex can be main­tained in spines for about 30 min after LTP induction,
longer than the bulk CaMKII activation. Thus, reciprocal activation within a kinase–effector complex may be a
general mechanism to maintain local CaMKII signallingfor an extended period.
ARC. Strong activation of neurons induces transcription of the immediate-early gene Arc. ARC accumulates
in non-stimulated spines, but is extruded from spines
undergoing LTP, and the levels of ARC are inversely
correlated with surface GluA1 expression70. The accumulation of ARC in non-stimulated spines requires
ARC–CaMKIIβ interaction, which is diminished byincreased Ca2+–CaM levels as occurs in stimulated
spines. Thus, the high calcium levels during LTP probably reduce CaMKIIβ–ARC interactions, reducing the
localization of ARC in stimulated spines.
As ARC can promote the endocytosis of AMPARs104,105,
this so-called inverse tagging of non-stimulated
spines with ARC may increase the contrast of synaptic
weights between stimulated spines that are undergoing
Hebbian LTP and the surrounding non-stimulated spinesthat experience a modest but general downscaling. Somework indicates that experience-dependent plasticity
induces this inverse tagging mechanism to sharpen
response tuning of cortical neurons110. Remarkably,
ARC forms a virus-like capsid that is transmitted between
cells111,112. Whether the inverse tagging requires assemblyof a capsid structure and its transmission, and whether
this transmission mediates downscaling of synapses thatsurround capsid-release sites as a potential mechanism ofdownscaling synapses formed by other, nearby neurons
are important questions for future study.

Molecular mechanisms of signalling
Role of kinase activity. Early evidence implicating
CaMKII in LTP was provided by postsynaptic expression or acute injection of a constitutively active mutant

Reviews
form of CaMKIIα called ‘tCaMKII’, which contains thetruncated amino acid sequence 1–290 and thus no regu­
latory region. Both approaches resulted in an increasein AMPAR-mediated excitatory postsynaptic potential(EPSC) amplitude and occlusion of LTP113,114. Althoughspine size correlates well with synaptic strength (reviewed
in ref.115) and typically increases during LTP48,62, overexpression of tCaMKII increased EPSC amplitude but not
spine size116. Further in-depth analysis of the effects ofexpression of various CaMKII mutants on spine size versus EPSC amplitude revealed a remarkable bifurcationof these two parameters at the level of CaMKII (ref.116).
Specifically, CaMKII binding to GluN2B can drive spine
enlargement without requiring its catalytic activity,
whereas an increase in postsynaptic AMPAR activity
requires catalytically active CaMKII but potentially notits binding to GluN2B, as discussed below.
CaMKII mutants that can bind to GluN2B andform dodecamers invariantly increase spine size. Suchmutants include the K42R–T286D double mutant,which combines a kinase-null mutation and an
autophosphorylation-mimetic mutation, mimicking
the active open conformation but without catalytic
activity116. These findings suggest that CaMKII acts as astructural protein to augment spine size independentlyof its catalytic activity, whereas its kinase activity mediates T286 autophosphorylation, which should prolong
binding to GluN2B in intact cells and thereby potentially spine enlargement. Whereas K42R–T286D double
mutant and also T286D single mutant CaMKII increased
spine size, they decreased EPSC amplitude116. By contrast, the T286D–T305A–T306A triple mutant increased
both spine size and EPSC amplitude116. These results
can be best explained by assuming that the T286D single mutant autophosphorylated itself on T305 or T306
even under basal, non-stimulated conditions117, whichblocked Ca2+–CaM binding and thereby full activation of
CaMKII (refs.50,55,118). The T286D–T305A–T306A combination of mutations prevented autophosphorylation of
T305 and T306 such that the kinase could be fully activated by Ca2+–CaM, resulting in increased EPSC amplitude. Thus, to increase EPSC amplitude, CaMKII has tobe able to be fully activated through Ca2+–CaM binding
to the autoinhibitory domain (or through the removal orabsence of the autoinhibitory domain, as in tCaMKII).
Nonetheless, an increase in EPSC amplitude wasobserved with T286D when it was expressed in the presence of the NMDAR antagonist APV116. Under these
conditions, calcium influx through the NMDAR shouldbe blocked, and so Ca2+–CaM would be expected not to beavailable to bind to the T305–T306 region to fully activate
CaMKII. This consideration raises the question of why
phosphorylation at T305 and T306, as presumably occurs
in the T286D single mutant, prevents an increase in EPSC
amplitude at all. A potential answer is that other sourcesof calcium could drive CaMKII activation under basal,
non-stimulated conditions to augment synaptic strength
upon expression of the T286D-mutant CaMKII, without
triggering phosphorylation of T305 or T306. For example,
LTP induced by a 5-Hz tetanus that is 3 min long withsimultaneous activation of the β2-adrenergic receptor
depended on calcium influx through the CaV1.2 channels

only and not at all through NMDARs119. However, this LTP
still also required CaMKII binding to GluN2B (H. Qianand J.W.H., unpublished results). Thus, blocking calcium influx through NMDARs seems to have prevented
phosphorylation at T305 and T306 of the T286D-mutant
CaMKII, enabling activation of this mutant in a mannerthat drives an increase in EPSC amplitude. Perhaps underbasal neuronal-activity conditions, NMDAR-mediatedcalcium influx is more effective than CaV1.2-mediatedinflux in stimulating T305/T306 phosphorylation and thus
preventing an increase in AMPAR activity. However,CaV1.2-mediated influx may be sufficient to enable temporary activation of CaMKII to drive the increase in
EPSC amplitude seen when T286D-mutant CaMKII isoverexpressed with APV present.
Single-cell, CRISPR-mediated knockout of the geneencoding CaMKIIα (which is three times more abundant than CaMKIIβ in the rat forebrain120), either alone
or in combination with knockout of the gene encoding
CaMKIIβ (double knockout) in the hippocampal CA1
region, reduces the AMPAR-mediated EPSCs by about
50% and NMDAR-mediated EPSCs by about 30%43.
These results imply that, under basal conditions (that is,with no specific treatment of the rodents or slices besides
routine handling), development of full synaptic strength
depends on CaMKII.
Furthermore, LTP is abolished in the CaMKIIαknockout and double-knockout neurons43. Knockoutof only CaMKIIβ also impairs LTP but not the basaltransmission of AMPAR-mediated EPSCs43. In doubleknockout neurons, restoration of expression of CaMKIIα
fully rescued AMPAR and NMDAR EPSCs and LTP,
whereas restoration of expression of CaMKIIβ rescued
only NMDAR-mediated EPSCs. Accordingly, CaMKIIα
is important for both basal postsynaptic AMPAR activity and LTP, whereas CaMKIIβ is dispensable for basal
AMPAR activity but is important for NMDAR function
and LTP. The specific roles of T286 phosphorylation, thekinase activity of CaMKII and the interaction of CaMKII
with GluN2B and other substrates were tested by overexpression of various mutant forms of CaMKIIα (including
the phospho-dead T286A and the kinase-dead K42R)
on the double-knockout background. This approachrevealed that both T286 phosphorylation and kinase
activity of CaMKIIα are necessary for rescuing AMPAR
current and LTP, but not for rescuing NMDAR current.
These results43 imply that a structural role of CaMKII
is sufficient to support the functional availability of
NMDARs at postsynaptic sites, whereas catalytic activity
is required to augment AMPAR current.
This requirement for catalytic activity to increase
AMPAR-mediated currents could reflect the need tophosphorylate other substrates, such as transmembrane
AMPAR regulatory proteins (TARPs; for example,
TARPγ8 (refs.88,121)) or RHO GTPase protein-activating
GEFs (such as kalirin 7 and TIAM1) that augment
F-actin formation and thereby spine growth to provide the structure for an increase in postsynaptic
AMPAR content (see later for discussion of RHO regu­
lation)44,122–124. Alternatively, CaMKII might need tomaintain its own phosphorylation of T286 for continued
interactions with other postsynaptic proteins, especiallywww.nature.com/nrn

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Reviews
Myristoylated
Bearing a lipid moiety known
as a myristoyl side chain.

GluN2B. Mutations at the extended substrate-binding
pocket (F98K and I205K; Box 1) that prevent CaMKII
binding to GluN2B and other CaMKAPs also impair
LTP and basal AMPAR current43. Thus, overall, T286
phosphorylation, kinase activity and substrate or
GluN2B binding are all required for the maintenance ofbasal AMPAR activity and LTP.
Why does single-cell knockout43 but not system-wide
knockout of CaMKIIα18 decrease AMPAR activity?
Also, single-point mutations that disable autophosphorylation (T286A in CaMKIIα)125 or kinase activity(K42R) do not change basal AMPAR-mediated synaptic function126 in respective knock-in mice, whereas
NMDAR-mediated transmission can be altered in theseanimals127. Furthermore, double knockout of CaMKIIαand CAMKIIβ in adult mice (8–10 weeks old) using a
Cre-dependent conditional knockout did not change
basal transmission, but completely abolished LTP128.
Perhaps CaMKII facilitates synaptic strengthening
through trans-synaptic mechanisms. Synapses in aknockout neuron could be at a collective disadvantage
and ‘lose out’ to other synapses formed nearby thatmight become stronger because resources of the presynaptic input neurons might be allocated to the synapses
that they form with surrounding neurons. Consistently,
presynaptic and postsynaptic parameters of synaptic
strength are strongly correlated (reviewed in ref.115),and overexpression of CaMKIIβ increases presynaptic
glutamate release probability, whereas its knockdown
prevents homeostatic synaptic upscaling induced bychronic inhibition of transmission129.
Pharmacological reversal of LTP and AMPAR basaltransmission. That basal AMPAR activity and LTP areboth affected by impaired CaMKII binding to GluN2B
supports the idea that, in vivo, LTP mechanisms maintain synaptic strength. This notion is supported byeffects of acute application of membrane-permeant,
CaMKII-inhibiting peptides, such as tatCN21, which
is derived from the CaMKII-inhibitory protein
CaMKIIN1130. Application of 5 µM tatCN21 blocked
CaMKII and induction of LTP, but this dose of tatCN21
did not reverse LTP when applied after LTP had alreadybeen induced 25. By contrast, application of 20 μM
tatCN21 displaced CaMKII from GluN2B, reversed LTP
and reduced basal synaptic transmission40, suggestingthat CaMKII–GluN2B binding increases postsynaptic
AMPAR activity.
myr-CN27, which expands the CaMKII-binding
sequence of tatCN21 by six residues, and myr-AIP2,
which is derived from the autoinhibitory domain of
CaMKII (refs.130,131), are two new peptides that share thesame binding site on CaMKII with tatCN21 but that are
myristoylated. The application of 1 μM myr-CN27 andmyr-AIP2 reduced basal transmission and reversed
LTP41. The simplest explanation for the different effectsof 5 µM tatCN21 versus 1 µM myr-CN27 and myr-AIP2
is that myr-CN27 and myr-AIP2 also displace CaMKII
from GluN2B with higher potency than does tatCN21.
Extensive controls for myr-CN21 and myr-AIP2 ruledout off-target presynaptic effects that have been reported
for tatCN21 (ref.41). Prolonged, but not brief, activation

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0123456789();:of the CaMKII inhibitor paAIP2 (10–50 μM estimatedintracellular concentration37) also reduced LTP maintenance and basal transmission41, further suggesting that
CaMKII–CaMKAP interactions are crucial for LTP
maintenance and basal transmission37.
Nanodomain signalling around the GluN2B–CaMKII
complex and synaptic plasticity. Imaging studies suggestthat CaMKII activation and accumulation are restricted
to those spines that experience potentiation 34,93.
Furthermore, CaMKII is necessary for standard LTP
in CA1, and LTP is impaired when CaMKII binding
to GluN2B is abrogated88. Thus, activity-dependent
CaMKII binding to GluN2B seems to be important forthe synapse specificity of LTP, a prerequisite for the roleof LTP in information storage, by recruiting CaMKII to
those synapses that undergo LTP.
How can GluN2B-associated CaMKII augment
the functional availability of AMPARs at postsynaptic
sites when localized at NMDAR nanodomains, which
only partially overlap with AMPAR nanodomains?132,133
NMDAR-anchored CaMKII might phosphorylate
nearby AMPARs that then become trapped when diffusing through AMPAR nanodomains. Consistent with this,activity-induced phosphorylation of GluA1 on S831 andpostsynaptic accumulation of auxiliary AMPAR TARP
subunits was impaired in knock-in mice that express
GluN2B L1298A–R1300Q and showed reduced CaMKII–
GluN2B binding88. S831 phosphorylation augments
single-channel conductance of AMPARs134,135, whichmight contribute to an increase in postsynaptic response
during LTP induction136. Alternatively, this increase inpostsynaptic response could be mediated by recruitment
of GluA1 homomeric AMPARs137 (see also ref.138), whichhave a higher conductance than do GluA2-containing
AMPARs139. However, LTP is normal in knock-in miceexpressing GluA1S831A, suggesting that S831 phosphorylation is not strictly necessary140. Also, S831 phosphorylation is not necessary for tCaMKII-induced translocationof GluA1 to the synapse141,142. Indeed, less than 1% of
GluA1 is phosphorylated, even after chemical LTP
induction143 (but see ref.142). Therefore, the relevance of
S831 phosphorylation is not fully clear.
A lasting increase in postsynaptic AMPAR function could be through phosphorylation of the auxiliary AMPAR subunit TARPγ2 (refs.144,145) or TARPγ8(ref.121) (Fig. 5). TARPs mediate postsynaptic localizationof AMPARs by binding to PSD95 and its homologues
PSD93 and synapse-associated protein 102 (SAP102;
also known as DLG3)145–148. TARP phosphorylation by
CaMKII augments TARP binding to PSD95 (refs.144,145),
trapping AMPARs at postsynaptic sites121,149 (but see
ref.150). However, further analysis is required to address
disparate findings that implicate CaMKII-mediatedphosphorylation of TARPγ2 (ref.151) versus TARPγ8(ref.121) in the hippocampus. The C-terminal intracellulardomains of TARPs contain clusters of positively charged
arginine residues (R225 to R250 in mouse TARPγ2, andR261 to R288 in TARPγ8), and multiple phosphorylated
serine residues have been identified in and around thisregion151. Two of these residues in TARPγ8 (S277 andS281) seem to be crucial CaMKII targets; although mice

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Ca2+

AMPAR

NMDAR

TARP

GluA1

GluN2B
CaMKII

P
S831

P
P
P
P

Detachmentof TARP
PSD95

Fig. 5 | role of caMKii binding to NMDA receptor subunit 2B in regulating AMPA
receptors. Calcium influx through NMDA receptors (NMDARs), as occurs duringlong-term potentiation, triggers binding of calcium–calmodulin (CaM)-dependent
kinase II (CaMKII) to NMDAR subunit 2B (GluN2B). At this location, CaMKII can effectively
phosphorylate nearby AMPA receptors (AMPARs) — at S831 in the GluA1 subunit of thereceptor — and their auxiliary subunits known as transmembrane AMPAR regulatory
proteins (TARPs). Phosphorylation of S831 of GluA1 increases AMPAR conductance.
Calcium also promotes detachment of the cytosolic carboxy termini (C termini) of TARPs,
which have multiple positively charged arginine and lysine residues, from the plasma
membrane. The detachment enables CaMKII to phosphorylate multiple residues in the
TARP C termini; phosphorylation of S277 and S281 in TARPγ8 has been specifically
implicated as important for long-term potentiation121. These phosphorylations reduce thenet positive charge of TARPγ8 and thereby reduce the reassociation of the TARP C terminiwith the plasma membrane. In this way, the number of TARP C termini that are available
for binding to PDZ domains of postsynaptic density protein 95 (PSD95) is increased,enabling more trapping of AMPAR–TARP complexes at postsynaptic locations149.

Consensus phosphorylationsites
Amino acid sequences that arerecognized by specific protein
kinases as substrate sites.

Half-maximal effective
concentration
The concentration of amolecule at which it exhibits
half-maximal efficacy withrespect to, for example,
enzymatic activity or binding
interactions.

expressing TARPγ8 with these residues replaced withalanine show normal basal synaptic transmission, theyshow impaired LTP, learning and memory121. Therefore,
CaMKII may mediate LTP partly by phosphorylating
TARPs (Fig. 5).
Under basal conditions, TARP C termini are associated with the plasma membrane, owing to electrostaticinteractions between their multiple positively charged
arginine residues (which are part of the CaMKII
consensus phosphorylation sites) and negatively charged
phospholipids144,145,152. Because calcium ions disrupt suchelectrostatic interactions, NMDAR-mediated calcium
influx may make TARPs more accessible for CaMKII,
by decreasing association of their C termini with theplasma membrane144,145,152. TARP phosphorylation by
CaMKII prevents reassociation of TARP C termini withthe plasma membrane and thus augments increases in
TARP–PSD95 binding144,145 and postsynaptic AMPAR
anchoring. Consistent with this notion, chemicallyinduced LTP increases the content of TARPγ8 and other
TARPs in PSD preparations88.

LTP is impaired in mice expressing S277A and S281A
double-mutant TARPγ8, but not when all endogenous AMPARs are replaced with a GluA1–TARPγ8fusion protein with these S277A/S281A mutations150.
Conversely, postsynaptic AMPAR localization is disrupted when the four C-terminal residues of TARP-γ8,
TTPL, which mediate PDZ binding, are removed fromthe GluA1–TARPγ8 fusion protein150 but not when they
are removed from endogenous TARPγ8 (ref.153). Perhaps
the C terminus of GluA1-fused TARPγ8 has a higher
propensity than the free TARPγ8 to detach from theplasma membrane and thus to bind to PSD95, independent of its CaMKII-mediated phosphorylation (Fig. 5).
That TTPL is not required for (non-fusion) TARPγ8
to localize AMPARs153 could be explained by a second,
electrostatic interaction site between TARPs and the first
PDZ of PSD95 (ref.154).
CaMKII also mediates LTD. CaMKII decreases postsynaptic AMPAR content by phosphorylating GluA1
on S567 (refs.10,155). LTD does not depend on CaMKII–
GluN2B binding88, which is prevented during LTD byanother Ca2+–CaM-dependent kinase, death-associatedprotein kinase 1 (DAPK1), whose binding site on
GluN2B overlaps with that of CaMKII (ref.156). DAPK1
blocks CaMKII binding during modest, LTD-driving
but not large, LTP-driving calcium influx156. In vitro,
increasing Ca2+–CaM concentrations cause a shift from
GluN2B–DAPK1 binding, which is impaired by Ca2+–
CaM, to GluN2B–CaMKII binding, which is induced
by Ca2+–CaM42,157 (at a half-maximal effective concentrationof about 300 nM (ref.156)). In addition, during LTD, thephosphatase calcineurin, which is activated at lower
Ca2+–CaM levels than CaMKII is, dephosphorylates
DAPK1 on S308, which promotes DAPK1 binding to
GluN2B156. However, mice that express a mutant version
of GluN2B that is deficient in CaMKII binding show
LTD that is insensitive to DAPK1 inhibition156, implyingthat other CaMKII-mediated mechanisms in addition to
DAPK1–CaMKII competition are at work during LTD,at least in these mice.
The modest but prolonged increase in Ca2+–CaM levels during LTD induction also promotes CaMKII autophosphorylation of T305 and T306, which is required for
LTD but not LTP51. This phosphorylation is slow in vitro
and more prominent during LTD-inducing prolonged
but low-frequency tetani than during LTP-inducing
short-duration but high-frequency tetani (100 Hz for
1 s), as it requires the slow unbinding of Ca2+–CaM
from CaMKII. The phosphorylation of T305 and T306
reduces CaMKII–NMDAR binding157 and increases
autonomous CaMKII activity51, which could especiallyapply to phosphorylation sites in CaMKII target proteins that are important for LTD. LTD-inducing but not
LTP-inducing stimuli also increase the phosphorylationof GluN2B on S1303 (in the GluN2B–CaMKII-bindingsite) by DAPK1 (ref.158), decreasing CaMKII–GluN2B
binding159,160. Accordingly, the modest but long-lasting
increase in Ca2+–CaM levels during LTD induction
can distinguish LTD from LTP by impairing CaMKII
binding to GluN2B through supporting the autophosphorylation of CaMKII on T305 and T306 and throughphosphorylation of GluN2B on S1303 by DAPK1.
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Reviews
Retromer complex
The complex formed by thevacuole protein sorting geneproducts VPS26, VPS29
and VPS35.

Spine pruning
Loss of spines or reduction inspine number over timein the brain, especially during
development but also laterin life.

Role of CaMKII in NMDAR-dependent metaplasticity.
LTP can induce a rapid switch (within 5 min) frommostly GluN2B-containing NMDARs to those containing GluN2A or GluN2A plus GluN2B at potentiatedpostsynaptic sites161. CaMKII phosphorylates GluN2A
on S1459, five residues upstream of its C terminus,
which constitutes a PDZ domain binding motif162,163.
S1459 phosphorylation of GluN2A augments the binding of sorting nexin 27 (SNX27) to the PDZ domain,and SNX27 in conjunction with the retromer complex
enhances surface expression of GluN2A duringchemically induced LTP162,163. This phosphorylation
also increases calcium influx through NMDARs163,
potentially creating a positive feedback loop to keep
CaMKII bound to GluN2B (as part of heterotrimeric
GluN1–GluN2A–GluN2B NMDARs139). In addition,
CaMKII recruits the casein kinase CK2 to GluN2B,
which phosphorylates on S1480 to impair PSD95 binding and surface expression of GluN2B164, potentiallyleading to a selective loss of GluN1–GluN2B heterodimeric NMDARs. Also, the switch from GluN2B to GluN2A
is reduced in CaMKIIα T286A knock-in mice, implicating prolonged CaMKII activity or CaMKII–GluN2B
binding in this process127.
Regulation of small GTPases and actin cytoskeleton.
F-actin is the main cytoskeletal component in spinesand governs spine size and LTP61,92,124,165. Formationof F-actin is controlled by the small GTPases CDC42,RHOA and RAC1, which are regulated by CaMKII(refs.44,124,165,166). Postnatal deletion of Cdc42 or Rac1
from excitatory neurons impairs LTP, sLTP and learning,
implicating the regulation of actin by these GTPases insynaptic plasticity124,167,168. CaMKII supports the releaseof brain-derived neurotrophic factor (BDNF) fromstimulated spines, and autocrine activation of the BDNF
receptor TRKB on the same spine stimulates CDC42
and RAC1 (refs.124,166). These RHO-family GTPases areactivated by GEFs and inactivated by GTPase-activating
proteins. The RAC-specific GEFs kalirin 7, TRIO andTIAM1 are important for sLTP and LTP, linking CaMKII
signalling and RAC activation44,123. Activated RAC stimulates the serine–threonine kinase PAK, which phosphorylates and activates LIM domain kinase 1 (LIMK1).
LIMK1, in turn, inactivates the actin-depolymerizing
protein cofilin by phosphorylating it at S3, leading to
the stabilization of F-actin in the spine head169. RAC
also couples to WAVE, which activates the actin-related
protein 2 (ARP2)–ARP3 complex, which nucleates andbranches F-actin filaments169.
CaMKII also regulates the small GTPase RAS
during LTP 170,171 by phosphorylating SYNGAP, a
RAS GTPase-activating protein concentrated in the
PSD172,173. SYNGAP phosphorylation triggers its dispersal from PSDs and spines, disinhibiting RAS174, which
in turn activates extracellular signal-regulated kinases(ERKs)170,175 and supports sLTP174. Notably, close relatives of RAS — RAP1 and RAP2 — have roles in LTD
and reversal of LTP (depotentiation)106,170,176. The signalling seems to occur in specific nanodomains, as
determined by dominant-negative mutants targeted todifferent subcellular domains106.

NaTure Reviews | Neuroscience
0123456789();:Whereas CDC42 shows spine-specific activation,
RAC1, RHOA and RAS signals spread out of the stimulated spine and into surrounding spines 5–15 µm away
(refs. 124,165,171,177) . This spread does not cause LTP or
enlargement in surrounding spines by itself, but instead
can facilitate LTP when combined with the stimulationof these spines124,165,171. Thus, LTP probably depends
on the concerted regulation of these GTPases, andactivation of a single one is insufficient124.

Photomanipulation of CaMKII signalling
Electrical or chemical stimulation activates manykinases in addition to CaMKII, complicating interpretation of the actions of CaMKII in studies using suchmanipulations. To resolve this problem, a photoactivatable CaMKII has been developed178. Blue light-activatedlight–oxygen–voltage-sensing domain 2 was fused tothe hinge region of CaMKII, inducing a catalytically
active open conformation of CaMKII, which triggers spine enlargement and LTP. Unlike for glutamate
uncaging-induced sLTP, this spine enlargement is protein synthesis dependent. Apparently, the stimulationof glutamate receptors renders LTP protein synthesis
independent through unknown mechanisms that are
not engaged by CaMKII activation alone.
Interestingly, photoactivatable CaMKII activatesspine CDC42, but not RHOA, although glutamate
uncaging leads to activation of RHOA via CaMKII.
Thus, RHOA activation in response to glutamate uncaging may require other calcium-dependent processes in
addition to CaMKII, which may indirectly regulate
RHOA. RHOA signalling is involved in spine pruning andshrinkage, whereas RAC1 and CDC42 promote spine
maturation169. Given that spine structure under basal
conditions is stable over days179, RHOA may destabilize spines so that they can undergo structural changes
during synaptic plasticity180.
To link the timescales of CaMKII action in synapses with the timescale of behaviour, it is necessary to
manipulate CaMKII in a temporally controlled manner.
Traditionally, this has been done using pharmacologicaltools. For example, in the contextual fear-conditioning
paradigm, when tatCN21, which crosses the blood–
brain barrier, is injected before conditioning, the memory tested 24 h later is impaired25. However, there isno effect on fear memory when tatCN21 is injected
immediately before the memory test. This suggests that
CaMKII activity is required for formation of this memory, but not for its maintenance or recall25. By contrast,
viral transduction of dominant-negative CaMKII after
establishment of conditioned place avoidance memory
extinguished this memory, suggesting a role of CaMKII
in the maintenance of this memory181. However, onemust bear in mind that such manipulations in vivo have
limited temporal resolution (hours for pharmacologicalcompounds, and days for viruses), making it difficult todistinguish between loss of synaptic plasticity and any
adaptations caused by the manipulation. Furthermore,
pharmacological tools are not cell type specific. Also, it isunclear whether the absence of an effect on fear-memory
recall by tatCN21 injection in vivo is perhaps because theconcentration of this peptide in the relevant neurons is

Reviews
Inhibitory avoidance task
Behavioural task in which
an animal is trained to avoid
a situation or localization.

1.

2.

3.
4.

5.

6.

7.

8.

9.

sufficient to inhibit CaMKII activity but not high enoughto displace CaMKII from GluN2B, with the latter action
but not former action being expected to reverse LTP40
and thereby impair memory.
By contrast, the photoinducible CaMKII inhibitor
paAIP2 enables determination of the timescale, cell
types and brain region of global CaMKII action for
specific behavioural adaptations, including learning and memory, with much higher temporal
resolution37,182,183. The temporal window of CaMKII
action required in excitatory neurons of the amygdala
for the inhibitory avoidance task was examined withuse of paAIP2. Memory formation in the task was
impaired when CaMKII was inhibited by illuminationof paAIP2-expressing neurons during, but not 3–5 minafter, training37. Accordingly, CaMKII activation during,
but not after, training is necessary to form avoidance
memory.
Synaptic plasticity in the motor cortex seems to be
crucial for motor skill learning. However, it has beendifficult to assess which neurons undergo the synaptic
plasticity that is relevant to motor skill learning. In one
study, mice were trained to walk on a moving treadmillusing only their front limbs, while their rear limbs were
fixed182. This behavioural plasticity required disinhibition of the activity of pyramidal neurons in the motor
cortex by somatostatin-positive (SST+) interneurons
during learning. When CaMKII activity was inhibited(with use of paAIP2) in the pyramidal neurons during
training, both circuit plasticity and learning were inhibited. These results suggest that CaMKII-dependent synaptic plasticity in pyramidal neurons is required for thisform of motor skill learning, and that SST+ interneurons
regulate the synaptic activity and, thereby, the synaptic
plasticity of those pyramidal neurons.
In Drosophila, paAIP2 was used to measure the roleof CaMKII in the timing of behavioural motivation183.
Accordingly, CaMKII activity in four male-specificneurons (Crz+ neurons) determines the duration of
copulation. Early in mating, the male persists througheven life-threatening stimuli, but his persistence declines

Lisman, J., Yasuda, R. & Raghavachari, S. Mechanismsof CaMKII action in long-term potentiation. Nat. Rev.
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over the course of 15–20 min, until even mild challenges
cause him to flee. Using heat as a life-threatening stimulus together with paAIP2, the authors measured thetemporal window of CaMKII activation during thismotivational shift. This approach revealed that CaMKII
is activated at the onset of mating, presumably by anincrease in intracellular calcium concentration. Also, theCamui FRET sensor revealed that CaMKII activity lasts
for about 6 min, much longer than the elevation in intracellular calcium concentration. Active CaMKII preventsthe motivational shift by life-threatening stimuli (suchas heat) during this time window. How CaMKII physiol­ogically affects Crz+ neurons is to be determined, butthese experiments suggest that calcium signalling may
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Conclusions and perspective
CaMKII received early attention in postsynaptic signalling because of its abundance, unusual structure andpresumed self-perpetuating autoregulation. Recent work
has defined potential key postsynaptic CaMKII substrates, including TARPs and RAC and RHO GEFs, andhas highlighted the importance of the role of CaMKII
as a structural protein that tunes itself through autophosphorylation towards binding various CaMKAPs.
However, much work lies ahead of us with respect todefining the molecular mechanisms of synaptic plasticity and learning. Central to such future work will be
the development of new tools, especially to define how
localizing CaMKII to nanodomains supports synaptic
plasticity. We anticipate the creation of tools that specifically allow targeting of CaMKII and reporters of its
activity to NMDARs and other postsynaptic anchoring
proteins for CaMKII to elucidate details of nanospace
signalling. Such new technologies will allow us to further
define the numerous and diverse functions of CaMKII
in health and disease.
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Acknowledgements
The authors thank K. U. Bayer and M. M. Stratton for criticalreading.

Author contributions
The authors contributed equally to all aspects of the article.

Competing interests
The authors declare no competing interests.

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Nature Reviews Neuroscience thanks N. Waxham and theother, anonymous, reviewer(s) for their contribution to
the peer review of this work.

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