Calmodulin binding sites can be defined for skeletal, cardiac, and brain ryanodine receptor (RYR) Ca2+ channels. Cardiac and brain RYR peptides corresponding to the calmodulin binding sites present in the skeletal RYR have been synthesized, and their interactions with calmodulin have been monitored. The central portions of the skeletal, cardiac, and brain RYR protomers display two calmodulin binding sites, one with high affinity and one with low affinity. Depending on the RYR model having 4 or 12 transmembrane segments, a third calmodulin binding site (CaM3) has been identified a few residues upstream from the putative transmembrane segment M1 or M5. Its affinity for calmodulin varies between the RYR isoforms: the cardiac RYR CaM3 displays a high CaM affinity, while the skeletal and brain RYR CaM3 both have low affinity, the lowest affinity being displayed by the brain isoform. The RYRs calmodulin binding site CaM1 encompasses the sequence Arg-His-Arg-Val(Ile)-Ser-Leu (PM1 peptides), which is phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase. Phosphorylation of RYR PM1 peptides occurs on the Ser, corresponding to amino acid number 2919, 3020, and 3055 of the brain, cardiac, and skeletal RYR protomers, respectively. Phosphorylation of the RYR PM1 peptides is inhibited by calmodulin binding, and the formation of the PM1 peptide-calmodulin complex is inhibited by peptide phosphorylation. These data indicate that the effect of calmodulin binding to RYR CaM1 may be regulated by the phosphorylation state of the Ser residue localized within the sequence Arg-His-Arg-Val(Ile)-Ser-Leu (Guerrini, 1995).

Fertilization of oocytes incites numerous changes relying on Ca2+ signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, this calcium channel has been shown to be asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FK-506 binding protein (FKBP) and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca2+ release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself (Albrieux, 2000).

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. In sea urchin eggs, Calmodulin acts as a functional mediator of cADPR-triggered CICR, through the RyRLC cADPR-induced Ca2+ release. This consists of two phases, an initial rapid release phase and a subsequent slower release. The second phase is selectively potentiated by calmodulin, which in turn, is activated by Ca2+ released during the initial phase. Caffeine enhances the action of calmodulin. Calmodulin does not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling (Tanaka, 1995).

The association of an endogenous, Ca(2+)-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) has been demonstrated. The activity of this thiol-protease is dependent on Ca2+ ion. These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356-1367 of the RyR. The cleavage site, the calmodulin binding site, is found at residues 1383-1400. The RyR cleavage by the Ca(2+)-dependent thiol-protease is prevented in the presence of ATP (1-5 mM) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. These data suggest a possible involvement for this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity (Shoshan-Barmatz, 1994).

Cisternae vesicles from rabbit skeletal muscle were fused into planar bilayers and the effect of calmodulin on single Ca2+ release channel (ryanodine receptor) currents was investigated. In the presence of 10(-7) and 10(-9) M free [Ca2+], nanomolar concentrations of calmodulin activates the channel by increasing the open probability of single-channel events in a dose dependent manner. The activatory effect of calmodulin was reversed by 10 microM ruthenium red. At high, 10(-5) M, free Ca2+ ion, calmodulin inhibits channel activity. Calmodulin overlays have been carried out using concentrations of Ca2+ ion similar to those used for the planar lipid bilayer assays. In the presence of 10(-7) M Ca2+ ion, calmodulin binds to the ryanodine receptor, to a region defined by residues 2937-3225 and 3546-3655. These results suggest that calmodulin may activate the Ca(2+)-release channel (ryanodine-receptor) by interacting with binding sites localized in the central portion of the RYR protomer (Buratti, 1995).

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors (see Drosophila InsP3R) were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly binds to multiple sites on InsP3 receptors and Ca2+ increases the binding by 190% +/- 10%; the half-maximal effect occurs when the Ca2+ concentration is 184 +/- 14 nM. In the absence of Ca2+, calmodulin causes a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect is similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibita the Ca2+ release from cerebellar microsomes evoked by submaximal (but not by maximal) concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways (Patel, 1997).

Cyclic nucleotide-gated (CNG) channels form a family of ion channels that are gated open by cAMP and cGMP. In photoreceptors and olfactory neurons, these channels serve as final targets for cGMP- and cAMP-signaling pathways that are activated by light and odorants, respectively. A functionally significant feature of CNG channels is their Ca2+ permeability. At physiological extracellular Ca2+ concentrations, Ca2+ carries a substantial fraction of the total current passing through CNG channels. Ca2+ entry through CNG channels is crucially important for both excitation and adaptation of vertebrate photoreceptors and olfactory neurons as Ca2+ controls the activity of several signaling enzymes, including the CNG channels themselves. Ca2+/calmodulin (CaM) attenuates the activity of rod and olfactory CNG channels by increasing their apparent K1/2 for cGMP and cAMP. This modulation is believed to serve as one of several Ca2+-mediated feedback mechanisms that terminate the electrical response and set the sensitivity of photoreceptor cells and olfactory neurons. The mechanism of this modulation has been examined using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity similar to the native channel. Using surface plasmon resonance spectroscopy, two unconventional CaM-binding sites (CaM1 and CaM2) were identified, one in each of the N- and the C-terminal regions of the beta-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of Ca2+ concentrations occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects (Weitz, 1998).

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy: previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Using site directed mutagenesis it has been shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. The IQ motif binds CaM and disruption of this binding activity prevents Ca2+ inhibition. It is proposed that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition (Qin, 1999).

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. A calcium-dependent interaction is reported between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. These results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity (Lee, 1999).

L-type Ca2+ channels support Ca2+ entry into cells; this event triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that self-regulate through their own activity. Among the many types of voltage-gated Ca2+ channels, L-type Ca2+ channels in particular display inactivation and facilitation, both of which are closely linked to the prior entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensive biophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif and a nearby consensus calmodulin-binding isoleucine-glutamine ('IQ') motif in the carboxy terminus of the alpha1C channel subunit have been implicated. Calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and the nature of the modulatory effect has been shown to depend on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removes Ca2+-dependent inactivation and unmasks a strong facilitation; conversion of the same residue to glutamate eliminates both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation (Zuhlke, 1999).

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a 'dominant-negative' manner. This demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family (Peterson, 1999).

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 Ca2+ influx through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca2+/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. CaMKII is shown to directly bind to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIalpha by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca2+ influx but also close to alpha-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 (Leonard, 1999).

Homologs of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca2+ entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP3 receptors (IP3Rs) for activation. Trp4 and other Trp proteins also interact with IP3Rs. The IP3R-binding domain also interacts with calmodulin (CaM) in a Ca2+-dependent manner with affinities ranging from 10 nM for Trp2 to 290 nM for Trp6. In addition, other binding sites for CaM and IP3Rs are present in the alpha but not the ß isoform of Trp4. In the presence of Ca2+, the Trp-IP3R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP3Rs inhibits the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 µM) but not a low (5 µM) concentration of the Trp-binding peptide of the IP3R. These data support the view that both CaM and IP3Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP3Rs differ for each Trp (Tang, 2001).

Mammalian homologs of Drosophila Trp form plasma membrane channels that mediate Ca2+ influx in response to activation of phospholipase C and internal Ca2+ store depletion. Human Trp3 is activated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and interacting domains, one on Trp and two on IP3R. Trp3 binds Ca2+-calmodulin (Ca2+/CaM) at a site that overlaps with the IP3R binding domain. Using patch-clamp recordings from inside-out patches, it has been shown that Trp3 has a high intrinsic activity that is suppressed by Ca2+/CaM under resting conditions. Trp3 is activated by the following: a Trp-binding peptide from IP3R that displaces CaM from Trp3, a myosin light chain kinase Ca2+/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca2+/CaM. It is concluded that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP3Rs (Zhang, 2001).

The mechanism involved in [Ca2+]i-dependent feedback inhibition of store-operated Ca2+ entry (SOCE) is not yet known. Expression of Ca2+-insensitive calmodulin (Mut-CaM) but not wild-type CaM increases SOCE and decreases its Ca2+-dependent inactivation. Expression of TrpC1 lacking C terminus aa 664-793 (TrpC1deltaC) also attenuates Ca2+-dependent inactivation of SOCE. CaM interacts with endogenous and expressed TrpC1 and with GST-TrpC1 C terminus but not with TrpC1deltaC. Two CaM binding domains, aa 715-749 and aa 758-793, were identified. Expression of TrpC1delta758-793 but not TrpC1delta715-749 mimics the effects of TrpCdelta1C and Mut-CaM on SOCE. These data demonstrate that CaM mediates Ca2+-dependent feedback inhibition of SOCE via binding to a domain in the C terminus of TrpC1. These findings reveal an integral role for TrpC1 in the regulation of SOCE. A key issue that remains to be resolved is whether TrpC1 itself forms the pore of SOCC (Singh, 2002).

Ryanodine receptor (RyR) activation by cyclic ADP-ribose (cADPR, a naturally occurring metabolite of NAD+) is followed by homologous desensitization. Though poorly understood, this 'switching off' process has provided a key experimental tool for determining the pathway through which cADPR mediates Ca2+ release. Moreover, desensitization is likely to play an important role in shaping the complexities of Ca2+ signaling involving cADPR, for example, localized release events and propagated waves. Using the sea urchin egg, a role has been unmasked for calmodulin, a component of the RyR complex and a key cofactor for cADPR activity, during RyR/cADPR desensitization. Recovery from desensitization in calmodulin-depleted purified endoplasmic reticulum (microsomes) is severely impaired compared to that in crude egg homogenates. An active, soluble factor, identified as calmodulin, is required to restore the capacity of microsomes to recover from desensitization. Calmodulin mediates recovery in a manner that tightly parallels its time course of association with the RyR. Conversely, direct measurement of calmodulin binding to microsomes reveals a loss of specific binding during cADPR, but not IP3, desensitization. These results support a mechanism in which cycles of calmodulin dissociation and reassociation to an endoplasmic reticulum protein, most likely the RyR itself, mediate RyR/cADPR desensitization and resensitization, respectively (Thomas, 2003).

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca²⁺ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. R- and N-type channels undergo Ca²⁺-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca²⁺ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels -- independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca²⁺ activity, respectively (Liang, 2003).

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation. This downregulation occurs through activation dependent proteolysis at a membrane-proximal site. Calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. These results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. It is thought that calmodulin is already bound to the L-selectin cytoplasmic tail in the resting cell and that removal of calmodulin through cell activation results in L-selectin shedding. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis (Kahn, 1998).

Deflection of the mechanically sensitive hair bundle atop a hair cell opens transduction channels, some of which subsequently reclose during a Ca2+-dependent adaptation process. Myosin I in the hair bundle is thought to mediate this adaptation; in the bullfrog's hair cell, the relevant isozyme may be the 119-kDa amphibian myosin I beta. Because this molecule resembles other forms of myosin I, it was hypothesized that calmodulin, a cytoplasmic receptor for Ca2+, regulates the ATPase activity of myosin. To investigate the possibility that calmodulin mediates Ca2+-dependent adaptation, calmodulin action was inhibited and the results were measured with two distinct assays. Calmodulin antagonists increase photolabeling of hair-bundle myosin I by nucleotides. In addition, when introduced into hair cells through recording electrodes, calmodulin antagonists abolish adaptation to sustained mechanical stimuli. This evidence indicates that calmodulin binds to and controls the activity of hair-bundle myosin I, the putative adaptation motor (Walker, 1996).

CA2+-regulated protein kinases play critical roles in long-term potentiation (LTP). To better understand the role of Ca2+/calmodulin (CaM) signaling pathways in synaptic transmission, Ca2+/CaM was injected into hippocampal CA1 neurons. Ca2+/CaM induces significant potentiation of excitatory synaptic responses, which is blocked by coinjection of a CaM-binding peptide and is not induced by injections of Ca2+ or CaM alone. Reciprocal experiments demonstrate that Ca2+/CaM-induced synaptic potentiation and tetanus-induced LTP occlude one another. Pseudosubstrate inhibitors or high-affinity substrates of CaMKII or PKC block Ca2/CaM-induced potentiation, indicating the requirement of CaMKII and PKC activities in synaptic potentiation. It is thought that postsynaptic levels of free Ca2+/CaM is a rate limiting factor and that functional cross-talk between Ca2+/CaM and PKC pathways occurs during the induction of LTP (Wang, J. H., 1995).

One form of Long-term depression (LTD) that has been observed in the hippocampus requires activation of postsynaptic NMDA (N-methyl-D-aspartate) receptors, a change in postsynaptic calcium concentration, and activation of postsynaptic serine/threonine protein phosphatase 1 (PP1) or 2A (PP2A). The mechanism by which PP1 or PP2A is regulated by synaptic activity is unclear because these protein phosphatases are not directly influenced by calcium concentration. LTD induction may require activation of a more complex protein phosphatase cascade consisting of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, its phosphoprotein substrate, inhibitor-1, and PP1. This hypothesis was tested using calcineurin inhibitors as well as different forms of inhibitor-1 loaded into postsynaptic cells. These results suggest a signaling pathway in which calcineurin dephosphorylates and inactivates inhibitor-1. This in turn increases PP1 activity and contributes to the generation of LTD (Mulkey, 1994).

Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is (1) a key determinant of network properties; (2) is involved in many physiological processes such as motor control, sound localization, or sensory adaptation, and (3) is critically dependent on cytosolic [Ca²⁺]. However, the underlying molecular mechanisms and the identity of the Ca²⁺ sensor/effector complexes involved are unclear. This study identifies 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 (Junge, 2004).

Neurons transfer information at chemical synapses. Interestingly, synaptic activity does not only transmit information but also regulates synaptic strength. Such activity-dependent modification of synaptic performance, or synaptic plasticity, is essential for information processing, learning, and memory (Junge, 2004).

Short-term synaptic plasticity (STP) occurs during and after repetitive synaptic activity on a timescale of milliseconds to minutes. It is a key determinant of network processes and is involved in brain functions as diverse as motor control, sensory adaptation, sound localization, and cortical gain control. STP can be expressed either as short-term enhancement (STE) or short-term depression (STD), depending on the initial release probability (P_r) of the synapses involved. High P_r is usually associated with STD, while a low P_r favors STE (Junge, 2004 and references therein).

Depletion of a readily releasable pool of fusion-competent synaptic vesicles (RRP) is a major cause for STD. The generation of this RRP is absolutely dependent on the priming action of UNC-13/Munc13s. The level of STD under steady-state conditions of RRP depletion and replenishment is controlled by a Ca²⁺-dependent vesicle supply process, of which the molecular mechanism and significance for STP are poorly understood. Calmodulin (CaM) may mediate this Ca²⁺-dependent process by acting on a subpool of the RRP with high P_r (Junge, 2004 and references therein).

A second well-known form of STP is STE. Three major forms of STE, facilitation, augmentation, and potentiation, can be distinguished based on their lifetime. During sustained activity, the efficacy of release is increased in STE, but it is unclear whether this is due to increased vesicular P_r or RRP size or both. STE is critically dependent on increased concentrations of residual Ca²⁺ ([Ca²⁺]_res), which accumulates during action potential activity due to incomplete elimination. According to the original residual Ca²⁺ hypothesis, [Ca²⁺]_res was thought to act on the secretory Ca²⁺ sensor. However, given the differences in Ca²⁺ requirements of fast neurotransmitter release and STE, additional, high-affinity Ca²⁺ sensors likely contribute to STE. The identification of such high-affinity Ca²⁺ sensors whose characteristics are compatible with the Ca²⁺ dynamics in presynaptic terminals and of molecules that transduce the residual Ca²⁺ signal to the secretory machinery during STE is essential for a mechanistic understanding of STE (Junge, 2004 and references therein).

The Munc13 proteins (Munc13-1, the splice isoforms bMunc13-2 and ubMunc13-2, and Munc13-3) are candidate mediators of STP. Genetic studies in mouse, fly, and nematode have established an essential role for this presynaptic protein family in synaptic vesicle priming and RRP generation. Munc13s regulate the SNARE protein Syntaxin and promote SNARE complex formation and fusion competence of synaptic vesicles (Junge, 2004 and references therein).

By determining synaptic vesicle priming, Munc13s modify synaptic strength. The domain structure of Munc13s with several binding sites for second messengers and regulatory proteins indicates that this function is tightly regulated. Indeed, Munc13s are targets of the diacylglycerol (DAG) second messenger pathway. The C₁ domain function of Munc13-1 is essential for DAG and phorbol ester (PE) binding and PE potentiation of synaptic amplitudes in hippocampal neurons. Moreover, rescue experiments in Munc13-1/2 double knockout (DKO) neurons show that STE is prevalent in neurons that express only ubMunc13-2, while moderate STD is prominent in neurons expressing only Munc13-1. Thus, Munc13 isoforms can differentially control STP, but the relation of this phenomenon to the long-established role of [Ca²⁺]_res in STP is unknown (Junge, 2004 and references therein).

This study reports that Munc13-1 and ubMunc13-2 bind CaM in a Ca²⁺-dependent manner via an evolutionarily conserved CaM recognition motif. Using synaptic depression, frequency facilitation, and augmentation protocols in autaptic hippocampal neurons (a special type of neuron that incorporates synaptical positive feedback through recurrent collaterals of its own axons) as a model of STP, it is shown that CaM binding to Munc13 proteins causes increased priming activity and RRP sizes. It is concluded that activation of the CaM/Munc13 complex by [Ca²⁺]_res represents a molecular correlate for the phenomenon of Ca²⁺-dependent vesicle pool refilling. This mechanism controls STP characteristics and is likely to be evolutionarily conserved (Junge, 2004).

Experience-dependent plasticity can be induced in the barrel cortex by removing all but one whisker, leading to potentiation of the neuronal response to the spared whisker. To determine whether this form of potentiation depends on synaptic plasticity, long-term potentiation (LTP) and sensory-evoked potentials were studied in the barrel cortex of alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII)T286A mutant mice. Three different forms of LTP induction were studied: theta-burst stimulation, spike pairing, and postsynaptic depolarization paired with low-frequency presynaptic stimulation. None of these protocols produced LTP in alphaCaMKIIT286A mutant mice, although all three were successful in wild-type mice. To study synaptic plasticity in vivo, measured sensory-evoked potentials were measured in the barrel cortex, and it was found that single-whisker experience selectively potentiates synaptic responses evoked by sensory stimulation of the spared whisker in wild types but not in alphaCaMKIIT286A mice. These results demonstrate that alphaCaMKII autophosphorylation is required for synaptic plasticity in the neocortex, whether induced by a variety of LTP induction paradigms or by manipulation of sensory experience, thereby strengthening the case that the two forms of plasticity are related (Hardingham, 2003).