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PubMed1994

Calmodulin-dependent enzymes undergo a protein-induced conformational change that is associated with their interactions with calmodulin.

The anionic hydrophobic (amphipathic) fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sulfonate was used to investigate the surface hydrophobic properties of calmodulin (CaM)-dependent enzymes as follows: calcineurin, myosin light chain kinase, cyclic nucleotide phosphodiesterase, CaM-dependent protein kinase II, and the gamma-subunit of phosphorylase kinase. We found that certain domains of these enzymes that interacted with 2-(p-toluidinyl)-naphthalene-6-sulfonate were exposed by a transient proton (H+) increase within the neutral pH range. This H(+)-induced exposure, which could be caused either by direct addition of H+ or by the release of H+ from metal chelators upon their binding of Ca2+, seemed to be more closely linked with a change in pH value (i.e. transient H+ increase) than with the actual equilibrium pH value of the system. Unlike the case with CaM-dependent enzymes, the H(+)-induced conformational change was uncommon in CaM-independent enzymes. When CaM-binding domains were removed from calcineurin and smooth muscle myosin light chain kinase, the resultant enzymes no longer exposed new domains in response to H+ increase. Using dansylated CaM to monitor the formation of CaM-enzyme complexes, we found that complex formation occurred with an uptake of H+ from solution. When CaM-dependent enzymes were evaluated at suboptimal concentrations of Ca2+, addition of H+ enhanced both the formation of CaM-enzyme complexes and the CaM-dependent catalytic activities, but this synergistic H+ effect occurred within only a narrow range of Ca2+ concentrations. These findings suggest that the H(+)-exposed domains in CaM-dependent enzymes are involved in the binding of CaM and that both conformational changes in CaM and its enzyme targets are necessary for complex formation. Further, the findings are consistent with the notion that CaM-binding domains are masked in the nonactivated (uncomplexed) conformations of CaM-dependent enzymes. The interplay between H+ and Ca2+ is discussed in relation to other systems that display interdependent effects of these two ions.

Animals↗
PubMed1994

The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.

The Na+/H+ exchange activity (NHE1 human isoform) is rapidly activated in response to growth factors and hyperosmotic stress. To get insight into the mechanism of NHE1 activation, we studied the direct interaction of a ubiquitous Ca(2+)-dependent regulatory factor, calmodulin (CaM) with NHE1. Binding experiments with CaM-Sepharose, as well as fluorescence measurements with dansylated CaM, revealed that the NHE1 cytoplasmic domain strongly binds CaM in a Ca(2+)-dependent manner. Fusion protein analysis with deletion mutants provided evidence for high (Kd approximately 20 nM) and intermediate (Kd approximately 350 nm) affinity CaM-binding sites located in neighboring regions of NHE1 (amino acids 636-656 and 657-700). To assess a regulatory role of CaM-binding sites, several cDNAs having deletion and point mutations in the high affinity site were generated and expressed in the exchanger-deficient fibroblast cell line PS120. Deletion and point mutations of positively charged residues of the high affinity CaM-binding site resulted in up to 50 and 80% reductions of cytoplasmic alkalinization caused by growth factors (alpha-thrombin, etc.) and 100 mM sucrose, respectively. In these mutants, the reduction in alkalinization was apparently in proportion to that of the CaM-binding ability. These results suggest that binding of Ca2+/CaM to the high affinity site is involved at least partly in the activation of NHE1 in response to different extracellular signals.

Amino Acid Sequence↗
PubMed1992

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.

Actomyosin↗
PubMed1987

Calmodulin-like proteins and communicating junctions. Electrical uncoupling of crayfish septate axons is inhibited by the calmodulin inhibitor W7 and is not affected by cyclic nucleotides.

The effects of W7, a calmodulin (CaM)-inhibitor, and cyclic nucleotides on electrical coupling and uncoupling are studied in crayfish lateral giant axons (septate axons). The septate axons provide a relatively simple two cell system in which both surface membrane and junctional resistance can be measured independently. Four microelectrodes are inserted into a septate axon, two on each side of the septum. Hyperpolarizing current pulses (150 nA) are injected alternatively in the caudal and rostral axon segment and the resulting electrotonic potentials are recorded. The axons are uncoupled at regular intervals by superfusing them with acetate-containing saline solution (pH 6.3) in the presence or absence of W7 (50-100 microM) or, as a control, its nonchlorinated form (W5). W7 strongly inhibits the acetate-induced increased in junctional resistance, while W5 is ineffective. The uncoupling inhibition does not appear to be caused by an increase in cyclic nucleotide concentration, because in preliminary experiments exposure to db-cAMP or db-cGMP (up to 1 mM) does not seem to influence either the basic values of Rj or their changes with acetate. The data confirm previous evidence for a participation of CaM-like proteins in cell-to-cell channel gating.

Animals↗
PubMed1995

UVB-induced calmodulin increase in pig epidermis: analysis of the effect of the calmodulin antagonist, W-13.

Calmodulin (CaM) is a multifunctional calcium-binding protein that has been implicated in the control of cell proliferation. In order to determine the role of CaM in keratinocyte proliferation, we investigated the effect of the CaM antagonist, W-13 on thymidine incorporation into pig epidermis. W-13 significantly inhibited thymidine incorporation into pig epidermis, while W-12, a closely related compound with much less anti-CaM activity, had little effect. The effect of W-13 was detected after as little as 2 h of incubation. Using a short-term (2-h) incubation system, the effects of other chemicals affecting various transmembrane signalling systems of keratinocytes were also investigated. None of these chemicals (epinephrine, histamine, forskolin, HA-1004, bradykinin, mezerein, phorbol 12-myristate, 13-acetate, H-7, staurosporin) inhibited thymidine incorporation. The effect of W-13 was reversible; its removal from the incubation medium resulted in the reinitiation of thymidine incorporation. Pig epidermis responded to 2.5 MED UVB irradiation showing an initial (24-48 h after irradiation) decrease and a subsequent (96-120 h after irradiation) increase in thymidine incorporation. The CaM content was not significantly altered during the initial hypoproliferative phase, but was significantly increased during the 72-120 h after UVB irradiation sometimes slightly preceding but mostly coinciding with the increase in thymidine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null.

Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca2+-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.

Amino Acid Sequence↗
PubMed1982

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin.

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.

Calcium-Binding Proteins↗
PubMedDate not supplied

Compartmentalization of calmodulin and tubulin in the male C57BL/6J mouse brain: heterogeneity of age changes in calmodulin compartments.

Calmodulin (CaM) and tubulin were analyzed by radioimmunoassay in subcellular fractions prepared from cerebral cortex and striatum of aging male C57BL/6J mice. Three fractions were prepared by a new procedure: cytosol (soluble); EGTA-releasable, membrane-bound; and detergent-extractable (Triton X-100), membrane-bound fractions. CaM concentration in all three fractions prepared from striatum showed small (10-15%, p less than 0.05) decreases with age (3-31 months). Cortical CaM concentrations were less affected by age, and only the EGTA-releasable fraction decreased. To compare functional activity and immunoreactivity of CaM, soluble CaM was also assayed by the activation of cyclic nucleotide phosphodiesterase (PDE). The radioimmunoassay and PDE activation assays gave equivalent results, suggesting that no alteration occurred with age in biological activity of CaM, via post-translational modification or other mechanisms. Soluble and particulate tubulin concentration did not change significantly with age in either brain region. The changes observed in striatal CaM, particularly in membrane-bound compartments, could contribute to the age-related decline in mammalian basal ganglial function.

Aging↗
PubMed1991

The naphthalenesulphonamide calmodulin antagonist W7 and its 5-iodo-1-C8 analogue inhibit potassium and calcium currents in NG108-15 neuroblastoma x glioma cells in a manner possibly unrelated to their antagonism of calmodulin.

Patch clamp techniques were used to record voltage-sensitive calcium and potassium currents from NG108-15 cells. N-(6-aminohexyl)-5-chloro-1-naphthalene- sulphonamide (W7), a calmodulin (CaM) antagonist and its more potent (10 times) 5-iodo-1-C8 analogue (J8) inhibited these currents in a dose-dependent manner. The inhibition was not dependent on internal or external Ca2+. W7 was about four times more potent as an inhibitor of the transient potassium current (IC50 = 8 microM) than of the M-current or of the calcium current. J8 was also selective for the potassium currents (IC50 values: transient current 4 microM, M-current 11 microM) compared to the calcium current (IC50 36 microM). It is suggested that the inhibition does not result from an anti-CaM action of the compounds.

Animals↗
PubMed2005

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin.

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.

Aluminum↗
PubMed1999

Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker.

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction.

Amino Acid Sequence↗
PubMed2003

Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

Androstadienes↗
PubMed1998

Alterations in calmodulin mRNA expression and calmodulin content in rat brain after repeated, intermittent amphetamine.

To assess whether calmodulin (CaM) gene expression could have a role in behavioral sensitization induced by repeated, intermittent amphetamine, CaM protein and mRNA of the three separate CaM genes were measured in several different brain areas from rats repeatedly administered saline or amphetamine. Rats were injected twice weekly for five weeks, followed by one week of withdrawal. CaM protein and mRNA were measured in dorsal striatum, limbic forebrain, prefrontal cortex, ventral mesencephalon and piriform cortex. There were increases of CaM protein content and decreases of CaM I mRNA in the dorsal striatum and prefrontal cortex. CaM II mRNA was also decreased in the dorsal striatum. A decrease of CaM protein and an increase of CaM I mRNA were found in the ventral mesencephalon. There was no change of CaM protein in the limbic forebrain, although a decrease of CaM I mRNA was detected. CaM protein and mRNA were not altered in the piriform cortex. Our findings demonstrate that both CaM content and mRNA are altered after an amphetamine regimen leading to sensitization. The fact that the changes in CaM content and mRNA are in dopaminergic brain areas associated with sensitization suggests that CaM could contribute to neurochemical events underlying behavioral sensitization to amphetamine.

Alternative Splicing↗
PubMed1999

The structural organization of the ascidian, Halocynthia roretzi, calmodulin genes. The vicissitude of introns during the evolution of calmodulin genes.

Two distinct calmodulin (CaM) genes are isolated from the ascidian, Halocynthia roretzi, (Hr-CaM A and Hr-CaM B) and those structures are determined. There are three nucleotide substitutions, producing two amino acid differences between Hr-CaM A and Hr-CaM B, and those are corresponding to two of the known eight variable residues among metazoan CaMs. Both Hr-CaM A and Hr-CaM B are constructed from six exons and five introns, and the positions of introns are identical. The positions of introns of Hr-CaMs are also identical with those of vertebrate CaMs, except third introns. The third introns of Hr-CaMs are inserted at 28bp upstream when compared with vertebrate CaMs. Thus, sliding of the third intron might have occurred in only the ascidian lineage prior to the gene duplication that also occurred only in that lineage. In addition, with the comparison of the intron positions, we attempt to investigate the vicissitude of introns during the evolution of metazoan CaMs.

Amino Acid Sequence↗
PubMed1996

A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch.

BACKGROUND: In contrast to conventional muscle myosins, where two different light chains (LCs) stabilize the elongated regulatory domain (RD) region of the head portion of the molecule, unconventional myosins are a diverse group of motors in which from one to six calmodulin (CaM) subunits are bound tandemly to the RD. In both cases, the heavy chains of the RDs have special sequences called "IQ motifs' to which the LCs or CaM bind. A previously puzzling aspect of certain unconventional myosins is their unusual mode of regulation, where activation of motility occurs at low levels of Ca2+. Although the atomic structure of the conventional muscle myosin RD has been determined, no crystallographic structure of the RD of an unconventional myosin is yet available. RESULTS: We have constructed a model of vertebrate CaM bound to the first IQ motif present in the neck region of an unconventional myosin (chicken brush border myosin I), using strict binding rules derived from the crystal structure of the scallop RD. The model accounts for aspects of the regulation of many unconventional myosins where CaM is bound at low levels of Ca2+ and released or changed in conformation at high levels of Ca2+. The conformational changes as a function of Ca2+ depend not only on the precise sequence of the IQ motifs but also on the interactions between CaM molecules bound to adjacent sites on the myosin heavy chain. CONCLUSIONS: According to our model, the full versatility of CaM binding to target peptides is displayed in the regulation of unconventional myosins. At low concentrations of Ca2+, CaM binds in a manner similar to the LCs of conventional myosins. At higher Ca2+ concentrations, CaM changes conformation and acts as a switch to regulate the activity of the unconventional myosin molecules.

Amino Acid Sequence↗
PubMed1993

Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae.

The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.

Amides↗
PubMed2003

Oxidation of Met144 and Met145 in calmodulin blocks calmodulin dependent activation of the plasma membrane Ca-ATPase.

Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met(144) and Met(145) or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50-60% reduction in the level of enzyme activation. Oxidation of Met(144) is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met(144) and Met(145) in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.

Amino Acid Sequence↗
PubMed2005

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).

2-Naphthylamine↗