Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Adhesion-dependent activation of CaMKII and regulation of ERK activation in vascular smooth muscle

Cell adhesion-dependent activation of ERK1/2 has been linked functionally to focal adhesion dynamics. We previously reported that in adherent vascular smooth muscle (VSM) cells, CaMKII mediates ERK1/2 activation in response to Ca(2+)-mobilizing stimuli. In the present study, we tested whether CaMKII regulates ERK1/2 signaling in response to VSM cell adhesion. Using an antibody that specifically recognizes CaMKII autophosphorylated on Thr(287), we determined that CaMKII is rapidly activated (within 1 min) after the adherence of cells on multiple ECM substrates. Activation of CaMKII on fibronectin was unaffected in cells overexpressing focal adhesion kinase (FAK)-related nonkinase (FRNK), an endogenous inhibitor of FAK. Furthermore, CaMKII was rapidly and robustly activated in VSM cells plated on poly-l-lysine. These results suggest that adhesion-dependent CaMKII activation is integrin independent. Adhesion-dependent FAK activation on fibronectin was not affected in cells treated with the selective CaMKII inhibitor KN-93 (30 muM) or in cells in which the expression of CaMKII with small interfering RNA (siRNA) was suppressed, although tyrosine phosphorylation of paxillin was inhibited in CaMKII-delta(2)-suppressed cells. Sustained ERK1/2 activation that was dependent on FAK activation (inhibited by FRNK) was also attenuated by CaMKII inhibition or siRNA-mediated gene silencing. Rapid ERK1/2 activation that preceded FAK and paxillin activation was detected upon VSM cell adhesion to poly-l-lysine, and this response was inhibited by CaMKII gene silencing. These results indicate that integrin-independent CaMKII activation is an early signal during VSM cell adhesion that positively modulates ERK1/2 signaling through FAK-dependent and FAK-independent mechanisms.     

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.     

Invited review: mechanisms of calcium handling in smooth muscles.

The concentration of cytoplasmic Ca(2+) regulates the contractile state of smooth muscle cells and tissues. Elevations in global cytoplasmic Ca(2+) resulting in contraction are accomplished by Ca(2+) entry and release from intracellular stores. Pathways for Ca(2+) entry include dihydropyridine-sensitive and -insensitive Ca(2+) channels and receptor and store-operated nonselective channels permeable to Ca(2+). Intracellular release from the sarcoplasmic reticulum (SR) is accomplished by ryanodine and inositol trisphosphate receptors. The impact of Ca(2+) entry and release on cytoplasmic concentration is modulated by Ca(2+) reuptake into the SR, uptake into mitochondria, and extrusion into the extracellular solution. Highly localized Ca(2+) transients (i.e., sparks and puffs) regulate ionic conductances in the plasma membrane, which can provide feedback to cell excitability and affect Ca(2+) entry. This short review describes the major transport mechanisms and compartments that are utilized for Ca(2+) handling in smooth muscles.     

CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca(2+) ](i) ) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca(2+) ](i) on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca(2+) with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca(2+) ](i) elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca(2+) ](i) elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca(2+) level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.     

Essential role for calcium waves in migration of human vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration is characterized by extension of the lamellipodia at the leading edge, lamellipodial attachment to substrate, and release of the rear (uropod) of the cell, all of which enable forward movement. However, little is known regarding the role of intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) in coordinating these distinct activities of migrating SMCs. The objective of our study was to determine whether regional changes of Ca(2+) orchestrate the migratory cycle in human vascular SMCs. We carried out Ca(2+) imaging using digital fluorescence microscopy of fura-2 loaded human smooth muscle cells. We found that motile SMCs exhibited Ca(2+) waves that characteristically swept from the rear of polarized cells toward the leading edge. Ca(2+) waves were less evident in nonpolarized, stationary cells, although acute stimulation of these SMCs with the agonists platelet-derived growth factor-BB or histamine could elicit transient rise of [Ca(2+)](i). To investigate a role for Ca(2+) waves in the migratory cycle, we loaded cells with the Ca(2+) chelator BAPTA, which abolished Ca(2+) waves and significantly reduced retraction, supporting a causal role for Ca(2+) in initiation of retraction. However, lamellipod motility was still evident in BAPTA-loaded cells. The incidence of Ca(2+) oscillations was reduced when Ca(2+) release from intracellular stores was disrupted with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin or by treatment with the inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxy-diphenyl borate or xestospongin C, implicating Ca(2+) stores in generation of waves. We conclude that Ca(2+) waves are essential for migration of human vascular SMCs and can encode cell polarity.     

P2X1 receptors mediate sympathetic postjunctional Ca2+ transients in mesenteric small arteries

Brief, spatially localized Ca(2+) transients occur in the smooth muscle adjacent to perivascular nerves of small arteries during neurogenic contractions. We named these "junctional Ca(2+) transients" (jCaTs) and postulated that they arose from Ca(2+) entering smooth muscle cells through P2X(1) receptors activated by neurally released ATP. Nevertheless, the lack of potent, subtype-selective P2X-receptor antagonists made determining the exact molecular identity of the channels difficult. Here we used small, pressurized mesenteric arteries from P2X(1)-receptor-deficient mice (KO) to test the hypothesis that jCaTs arise from Ca(2+) entering the smooth muscle cell via P2X(1) receptors. In wild-type (WT) arteries, confocal microscopy of fluo-4 fluorescence during electrical field stimulation (EFS) of perivascular sympathetic nerves revealed jCaTs in the smooth muscle cells adjacent to the perivascular nerves, similar to those reported previously in rat arteries, and alpha-latrotoxin (2.5 nM) markedly increased the frequency of "spontaneous" jCaTs. In the KO arteries, however, neither EFS nor alpha-latrotoxin elicited any jCaTs. A potent P2X-receptor agonist, alpha,beta-methylene ATP (10.0 microM), elicited strong contractions and increased intracellular Ca(2+) concentration in WT arteries but elicited neither in KO arteries. A biphasic vasoconstriction in response to EFS was observed in WT arteries. In KO arteries, however, the initial rapid, transient component of the biphasic vasoconstriction was absent. The data support the hypothesis that jCaTs represent Ca(2+) that enters the smooth muscle cells through P2X(1) receptors activated by neurally released ATP and that this Ca(2+) is involved in the initial rapid component of the sympathetic neurogenic contraction.     

Acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers stimulates glucose uptake by modulating Ca2+/calmodulin-dependent protein kinase II

Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is available regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intracellular Ca(2+) release (Ca(i)(2+)) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca(i)(2+) with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160 kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indicate that CLA isomers require Ca(i)(2+)-CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reactive oxygen species but had no effect on stress-activated protein kinase/c-jun NH(2)-terminal kinase. These data establish that t10,c12-CLA acts via Ca(i)(2+)-CaMKII-AMPK-AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glucose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition.     

Calcineurin Aalpha but not Abeta augments ICl(Ca) in rabbit pulmonary artery smooth muscle cells

Activation of Ca(2+)-dependent Cl(-) currents (I(Cl(Ca))) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca(2+)-dependent phosphorylation suppresses I(Cl(Ca)) in pulmonary artery myocytes, and the aim of the present study was to determine the role of the Ca(2+)-dependent phosphatase calcineurin on chloride channel activity. Immunocytochemical and Western blot studies with isoform-specific antibodies revealed that the alpha and beta forms of the CaN catalytic subunit are expressed in PA cells but that only the alpha variant translocated to the cell periphery upon a rise in intracellular [Ca(2+)]. I(Cl(Ca)) evoked by pipette solutions containing a [Ca(2+)] set at 500 nm was considerably larger when the pipette solution included constitutively active CaN containing the alpha catalytic isoform. This stimulatory effect was lost by boiling the enzyme or by the inclusion of a specific CaN inhibitory peptide and was not shared by the inclusion of the beta form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed I(Cl(Ca)) evoked by 500 nm Ca(2+) when the current amplitude was relatively large but was ineffective in cells with smaller currents. In perforated patch recordings, cyclosporin A consistently inhibited I(Cl(Ca)) evoked as a consequence of Ca(2+) influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of I(Cl(Ca)) is enhanced by Ca(2+)-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific.     

P2Y receptor subtypes evoke different Ca2+ signals in cultured aortic smooth muscle cells

Adenine and uridine nucleotides evoke Ca(2+) signals via four subtypes of P2Y receptor in cultured aortic smooth muscle cells, but the mechanisms underlying the different patterns of these Ca(2+) signals are unresolved. Cytosolic Ca(2+) signals were recorded from single cells and populations of cultured rat aortic smooth muscle cells, loaded with a fluorescent Ca(2+) indicator and stimulated with agonists that allow subtype-selective activation of P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Activation of P2Y1, P2Y2, and P2Y6 receptors caused homologous desensitisation, while activation of P2Y2 receptors also caused heterologous desensitisation of the other subtypes. The Ca(2+) signals evoked by each P2Y receptor subtype required activation of phospholipase C and release of Ca(2+) from intracellular stores via inositol 1,4,5-trisphosphate (IP(3)) receptors, but they were unaffected by inhibition of ryanodine or nicotinic acid adenine dinucleotide phosphate (NAADP) receptors. Sustained Ca(2+) signals were independent of the Na(+)/Ca(2+) exchanger and were probably mediated by store-operated Ca(2+) entry. Analyses of single cells established that most cells express P2Y2 receptors and at least two other P2Y receptor subtypes. We conclude that four P2Y receptor subtypes evoke Ca(2+) signals in cultured aortic smooth muscle cells using the same intracellular (IP(3) receptors) and Ca(2+) entry pathways (store-operated Ca(2+) entry). Different rates of homologous desensitisation and different levels of receptor expression account for the different patterns of Ca(2+) signal evoked by each P2Y receptor subtype.     

Selected contribution: roles of focal adhesion kinase and paxillin in the mechanosensitive regulation of myosin phosphorylation in smooth muscle.

The increase in intracellular Ca(2+) and myosin light chain (MLC) phosphorylation in response to the contractile activation of tracheal smooth muscle is greater at longer muscle lengths (21). However, MLC phosphorylation can also be stimulated by Ca(2+)-insensitive signaling pathways (19). The cytoskeletal proteins paxillin and focal adhesion kinase (FAK) mediate a Ca(2+)-independent length-sensitive signaling pathway in tracheal smooth muscle (30). We used alpha-toxin-permeabilized tracheal smooth muscle strips to determine whether the length sensitivity of MLC phosphorylation can be regulated by a Ca(2+)-insensitive signaling pathway and whether the length sensitivity of active tension depends on the length sensitivity of myosin activation. Although active tension remained length sensitive, ACh-induced MLC phosphorylation was the same at optimal muscle length (L(o)) and 0.5 L(o) when intracellular Ca(2+) was maintained at pCa 7. MLC phosphorylation was also the same at L(o) and 0.5 L(o) in strips stimulated with 10 microM Ca(2+). In contrast, the Ca(2+)-insensitive tyrosine phosphorylation of FAK and paxillin stimulated by ACh was higher at L(o) than at 0.5 L(o). We conclude that the length-sensitivity of MLC phosphorylation depends on length-dependent changes in intracellular Ca(2+) but that length-dependent changes in MLC phosphorylation are not the primary mechanism for the length sensitivity of active tension.     

Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.     

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.     

Involvement of ryanodine receptors in pacemaker Ca2+ oscillation in murine gastric ICC

Using a cell cluster preparation from the stomach smooth muscle tissue of mice, we measured intracellular Ca(2+) oscillations in interstitial cells of Cajal (ICCs) in the presence of nifedipine. Pacemaker [Ca(2+)](i) activity in ICCs was significantly suppressed by caffeine application and restored after washout. Application of either ryanodine or FK-506 terminated the pacemaker [Ca(2+)](i) activity irreversibly. Immunostaining of smooth muscle tissue showed that c-Kit-immunopositive cells (that form network-like structure cells in the myenteric plexus, equivalent to ICCs) clearly express ryanodine receptors (RyR). RT-PCR revealed that ICCs (identified with c-Kit-immunoreactivity) predominantly express type 3 RyR (RyR3). Furthermore, the FK-binding proteins 12 and 12.6, both of which would interact with RyR3, were detected. In conclusion, we provide first evidence for the essential contribution of RyR to generating pacemaker activity in gastric motility. Similar mechanisms might account for spontaneous rhythmicity seen in smooth muscle tissues distributed in the autonomic nervous system.     

Dual mechanism of a natural CaMKII inhibitor

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function.     

CaMKII inactivation by extracellular Ca(2+) depletion in dorsal root ganglion neurons

A mechanism by which Ca(2+)/CaM-dependent protein kinase (CaMKII) is autophosphorylated by changes in extracellular calcium in the absence of detectable changes in cytoplasmic [Ca(2+)] has been identified. We find that when the external Ca(2+) concentration ([Ca(2+)](O)) is lowered, Ca(2+) is released from intracellular stores to maintain a constant cytoplasmic Ca(2+) level, gradually depleting the endoplasmic Ca(2+) stores. Accompanying the store-depletion is a rapid decrease in CaMKII activity. Approximately 25% of the measured CaMKII autophosphorylation in DRG neurons in culture can be regulated by Ca(2+) flux from intracellular stores caused by manipulating [Ca(2+)](O), as shown by blocking refilling of store-operated Ca(2+)-channels with SK&F 96365, Ruthenium Red, and a partial block with Ni(2+). Blocking voltage-gated Ca(2+)-channels with either isradipine or SR 33805, had no effect on CaMKII autophosphorylation induced by restoring Ca(2+)(O) to normal after depleting the intracellular Ca(2+) stores. These results show that removal of Ca(2+)(O) has profound effects on intracellular Ca(2+) signaling and CaMKII autophosphorylation, in the absence of measurable changes in intracellular Ca(2+). These findings have wide-ranging significance, because [Ca(2+)](O) is manipulated in many experimental studies. Moreover, this explanation for the paradoxical changes in CaMKII phosphorylation in response to manipulating [Ca(2+)](O) provides a possible mechanism linking activity-dependent depletion of Ca(2+) from the synaptic cleft to a protein kinase regulating many neuronal properties.