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PubMed2005

Calmodulin regulation and identification of calmodulin binding region of type-3 ryanodine receptor calcium release channel.

Ryanodine receptors (RyRs) are a family of intracellular Ca(2+) channels that are regulated by calmodulin (CaM). At low Ca(2+) concentrations (<1 microM), CaM activates RyR1 and RyR3 and inhibits RyR2. At elevated Ca(2+) concentrations (>1 microM), CaM inhibits all three RyR isoforms. Here we report that the regulation of recombinant RyR3 by CaM is sensitive to redox regulation. RyR3 in the presence of reduced glutathione binds CaM with 10-15-fold higher affinity, at low and high Ca(2+) concentrations, compared to in the presence of oxidized glutathione. However, compared to RyR1 assayed at low Ca(2+) concentrations under both reducing and oxidizing conditions, CaM binds RyR3 with reduced affinity but activates RyR3 to a greater extent. Under reducing conditions, RyR1 and RyR3 activities are inhibited with a similar affinity at [Ca(2+)] > 1 microM. Mutagenesis studies demonstrate that RyR3 contains a single conserved CaM binding site. Corresponding amino acid substitutions in the CaM binding site differentially affect CaM binding and CaM regulation of RyR3 and those of the two other isoforms. The results support the suggestion that other isoform dependent regions have a major role in the regulation of RyRs by CaM [Yamaguchi et al. (2004) J. Biol. Chem. 279, 36433-36439].

Amino Acid Sequence↗
PubMed1996

Oxidative modification of a carboxyl-terminal vicinal methionine in calmodulin by hydrogen peroxide inhibits calmodulin-dependent activation of the plasma membrane Ca-ATPase.

In order to investigate the possibility that calmodulin (CaM) may be a principal target of reactive oxygen species (ROS) produced under conditions of oxidative stress, we have examined wheat germ CaM for the presence of highly reactive sites that correlate with the loss of function. Using reversed-phase HPLC and FAB mass spectrometry after proteolytic digestion, we have identified the sites of modification by hydrogen peroxide. We find that one of the vicinal methionines (i.e., Met146 or Met147) near the C-terminus of CaM is selectively oxidized. The ability of CaM to bind and to activate the plasma membrane (PM)-Ca-ATPase from erythrocytes was measured. There is a 30-fold decrease in the calcium affinity of oxidatively modified CaM. While there is a little change in the binding constant between the carboxyl-terminal domain of calcium-saturated CaM and a peptide homologous to the autoinhibitory sequence of the PM-Ca-ATPase, we find that there is a 9-fold reduction in the affinity of the amino-terminal domain of CaM with respect to the ability to bind target peptides. The extent of oxidative modification to one of the vicinal methionines near the carboxyl-terminal domain correlates with the loss of CaM-dependent activation of the PM-Ca-ATPase. The presence of oxidatively modified CaM prevents native CaM from activating the PM-Ca-ATPase, indicating that the oxidatively modified CaM binds to the autoinhibitory sequence on the Ca-ATPase in an altered nonproductive conformation. We suggest that the functional sensitivity of CaM to the oxidation of one of the C-terminal vicinal methionines permits CAM to serve a regulatory role in modulating cellular metabolism under conditions of oxidative stress. The predominant oxidation of a methionine near the carboxyl terminal of CaM is rationalized in terms of the enhanced solvent accessibility of these vicinal methionines.

Amino Acid Sequence↗
PubMed2000

Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?

The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells. To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit. We found a novel interaction between the C-terminal domain and calmodulin (CaM). By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-like and Baa motifs. Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca(2+). Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay. Although the NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-dependent spectral changes not only for NaBaa.CaM but also for NaIQ.CaM. The results, taken together with other results, indicate that whereas the NaBaa.CaM complex is formed in a Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.

Amino Acid Sequence↗
PubMed1997

In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase.

High molecular weight calmodulin binding protein (HMWCaMBP) is one of the major proteins expressed in bovine cardiac muscle. In this study, we report the phosphorylation and dephosphorylation of HMWCaMBP in vitro with a view to understand the function of this protein. The HMWCaMBP was phosphorylated by cAMP-dependent protein kinase with the incorporation of 2.30 mol of phosphate/mol of protein in the presence of EGTA. When phosphorylation was carried out in the presence of Ca2+/calmodulin (CaM), the incorporation of phosphate was reduced to 1.40 mol of phosphate/mol of protein. The decrease in the stoichometry of phosphorylation by Ca2+/CaM appears to be substrate directed i.e. due to the interaction of Ca2+/CaM with HMWCaMBP. The phosphorylated HMWCaMBP was unable to compete for free CaM in a CaM-dependent cyclic nucleotide phosphodiesterase (CaMPDE) assay. These results suggest that the phosphorylation sites may reside in or in proximity to the CaM-binding domain on HMWCaMBP since phosphorylated HMWCaMBP did not inhibit CaMPDE activity. HMWCaMBP was dephosphorylated by CaM-dependent phosphatase, calcineurin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7.

We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.

Adenocarcinoma↗
PubMed1993

Ca(2+)-calmodulin-dependent modification of smooth-muscle myosin light-chain kinase leading to its co-operative activation by calmodulin.

It has recently been shown that at relatively high molar ratios of myosin light-chain kinase (MLCKase) to calmodulin (CM) almost complete inhibition of the kinase activity occurs [Sobieszek (1991) J. Mol. Biol. 220, 947-957]. This inhibition resulted in a highly co-operative activation of MLCKase by CM, whereas the opposite activation (of CM by kinase) was hyperbolic, as expected (unco-operative). This difference in activation was observed only for kinase preparations preincubated with sub-stoichiometric amounts of CM, and only when micromolar concentrations of Ca2+ were present. The inhibitory effect was variable and depended not only on the concentration ratio of kinase to CM but also on the MLCKase preparation. For most of the preparations full inhibition required 5-15 min of preincubation at 25 degrees C and a 3-6-fold molar excess of kinase over CM. The inhibition was reversible, since full activity could be obtained after saturation of the kinase by additional CM. The inhibitory effect did not require ATP (excluding phosphorylation-type modifications of the kinase), and dephosphorylation of the kinase was not involved, since inhibition of an endogenous MLCK phosphatase by microcystin-LR did not decrease the inhibitory effect. Since the co-operative activation by CM was observed for cross-linked MLCKase preparations enriched in kinase dimers, but was absent for the analogous preparations enriched in the oligomers, we concluded that Ca(2+)-CM-dependent changes in the oligomeric state of the kinase were responsible for the modification observed. The exact nature of these modifications remains to be established.

Adenosine Triphosphate↗
PubMed2000

The calmodulin multigene family as a unique case of genetic redundancy: multiple levels of regulation to provide spatial and temporal control of calmodulin pools?

Calmodulin (CaM) is a ubiquitous, highly conserved calcium sensor protein involved in the regulation of a wide variety of cellular events. In vertebrates, an identical CaM protein is encoded by a family of non-allelic genes, raising questions concerning the evolutionary pressure responsible for the maintenance of this apparently redundant family. Here we review the evidence that the control of the spatial and temporal availability of CaM may require multiple regulatory levels to ensure the proper localization, maintenance and size of intracellular CaM pools. Differential transcription of the CaM genes provides one level of regulation to meet tissue-specific, developmental and cell-specific needs for altered CaM levels. Post-transcriptional regulation occurs at the level of mRNA stability, perhaps dependent on alternative polyadenylation and differences in the untranslated sequences of the multiple gene transcripts. Recent evidence indicates that trafficking of specific CaM mRNAs may occur to specialized cellular locales such as the dendrites of neurons. This could allow local CaM synthesis and thereby help generate local pools of CaM. Local CaM activity may be further regulated by post-translational mechanisms such as phosphorylation or storage of CaM in a 'masked' form. The spatial resolution of CaM activity is enhanced by the limited free diffusion of CaM combined with differential affinity for and availability of target proteins. Preserving multiple CaM genes with divergent noncoding sequences may be necessary in complex organisms to ensure that the many CaM-dependent processes occur with the requisite spatial and temporal resolution. Transgenic mouse models and studies on mice carrying single and double gene 'knockouts' promise to shed further light on the role of specificity versus redundancy in the evolutionary maintenance of the vertebrate CaM multigene family.

Animals↗
PubMed2001

Calmodulin priming: nuclear translocation of a calmodulin complex and the memory of prior neuronal activity.

The neuronal nucleus plays a vital role in information processing, but whether it supports computational functions such as paired-pulse facilitation, comparable to synapses, is unclear. Ca(2+)-dependent movement of calmodulin (CaM) to the nucleus is highly responsive to Ca(2+) entry through L-type channels and promotes activation of the transcription factor CREB (cAMP-responsive element binding protein) through phosphorylation by CaM-sensitive kinases. We characterized key features of this CaM translocation and its possible role in facilitation of nuclear signaling. Nuclear CaM was elevated within 15 s of stimulus onset, preceding the first signs of CREB phosphorylation in hippocampal pyramidal neurons. Depolarization-induced elevation of nuclear CaM also was observed in cerebellar granule cells, neocortical neurons, and dentate gyrus granule cells. Nuclear translocation of CaM was not blocked by disruption of actin filaments or microtubules, or by emptying endoplasmic reticulum Ca(2+) stores with thapsigargin. Translocation of fluorescently tagged CaM was prevented by fusing it with the Ca(2+)/CaM binding peptide M13, suggesting that nuclear CaM accumulation depends on association with endogenous Ca(2+)/CaM binding proteins. To determine whether increased nuclear [CaM] might influence subsequent nuclear signal processing, we compared responses to two consecutive depolarizing stimuli. After a weak "priming" stimulus that caused CaM translocation, CREB phosphorylation caused by a subsequent stimulus was significantly faster, more sensitive to Ca(2+) elevation, and less specifically dependent on Ca(2+) influx through L-type channels. CaM translocation not only supports rapid signaling to the nucleus, but also could provide a "memory" for facilitatory effects of repeated neural activity, seen in altered phosphorylated CREB dynamics and Ca(2+) channel dependence.

Animals↗
PubMed1999

Hormone-induced secretory and nuclear translocation of calmodulin: oscillations of calmodulin concentration with the nucleus as an integrator.

Many important enzyme activities are regulated by Ca2+-dependent interactions with calmodulin (CaM). Some of the most important targets for CaM action are in the nucleus, and Ca2+-dependent CaM translocation into this organelle has been reported. Hormone-evoked cytosolic Ca2+ signals occur physiologically as oscillations, but, so far, oscillations in CaM concentration have not been described. We loaded fluorescent-labeled CaM into pancreatic acinar cells and monitored the fluorescence in various regions by confocal microscopy. Sustained high concentrations of the hormone cholecystokinin or the neurotransmitter acetylcholine evoked a transient movement of cytosolic CaM from the basal nonnuclear area into the secretory granule region and, thereafter, a more substantial and prolonged translocation of CaM into the nucleoplasm. About 50% of the CaM that bound Ca2+ translocated. At a lower hormone concentration, evoking Ca2+ oscillations, regular spikes of increased CaM concentration were seen in the secretory granule region with mirror image spikes of decreased CaM concentration in the basal nonnuclear region. The nucleus was able to integrate the Ca2+ spike-evoked pulses of CaM translocation into a sustained elevation of the nucleoplasmic concentration of this protein.

Acetylcholine↗
PubMed1995

Identification of inhibitory and calmodulin-binding domains of the PDE1A1 and PDE1A2 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca2+/CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (KCaM) of recombinant PDE1A1 (0.3 nM) was approximately 10-fold less than the KCaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide that was still activated 3-fold by CaM (KCaM approximately 3 nM). However, complete CaM-independent activation occurred when residues 4-98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76-149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114-137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca2+ concentrations is located between the two CaM-binding domains.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Inducible nitric oxide synthase requires both the canonical calmodulin-binding domain and additional sequences in order to bind calmodulin and produce nitric oxide in the absence of free Ca2+.

All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca2+ in resting cells and when almost all Ca2+ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca2+ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca2+ to bind CaM and generate NO. In cell lysates, the concentration of Ca2+ necessary for half-maximal activity (EC50) was approximately 0 for NOS2, 200-300 n for NOS1, and 7-10 n for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC50, 200-300 n free Ca2+). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca2+-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca2+ levels approach zero.

Amino Acid Sequence↗
PubMed1998

Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. A novel calmodulin-peptide interaction motif.

The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an alpha-helix. The CaM-binding domain in the C-terminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an alpha-helical structure. NMR studies of biosynthetically [methyl-13C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N- and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed.

Amino Acid Sequence↗
PubMed2000

Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination.

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

Acetylcysteine↗
PubMed1985

Crystallization of calcium-calmodulin-trifluoperazine complex and an attempt at crystallizing calcium-free calmodulin.

Crystals suitable for X-ray structure analysis were obtained for CaM complexed with both calcium ions and a phenothiazine drug, trifluoperazine (TFP). The TFP/CaM binding ratio in crystals was experimentally determined to be nearly 1. An attempt at crystallizing calcium-free calmodulin (CaM) resulted in rigid but non-birefringent solids which exhibited no X-ray reflections.

Animals↗
PubMed2000

Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70.

Using a gel-overlay technique of biotinylated calmodulin (CaM), we showed that maize cytosolic Hsp70 protein could bind to CaM in the presence of 1 mM CaCl2. The purified maize cytosolic Hsp70 inhibited the activity of CaM-dependent NADK in a concentration-dependent manner. A synthetic peptide, which possesses the 21 amino acid sequence, PRALRRLRTACERAKRTLSST, at positions 261-281 in maize cytosolic Hsp70, could associate with CaM in the presence of 1 mM calcium. The synthetic peptide inhibited CaM-dependent NADK activity and PDE activity. This indicates that the 21-amino acid sequence at positions 261-281 is the CaM-binding site. The binding of CaM to Hsp70 inhibited the ATPase activity of Hsp70. The possible regulator function of Hsp70 in cell signaling events in response to heat stress is discussed.

Amino Acid Sequence↗
PubMed2004

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila.

A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM.

Animals↗
PubMed1996

Effect of calmodulin antagonists on calmodulin-induced biphasic modulation of Ca(2+)-induced Ca2+ release.

1. Calmodulin (CaM) has a biphasic effect on Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR): potentiation and inhibition at low (pCa > 6.0) and high (pCa 5) Ca2+ concentrations, respectively. To characterize the mode of action of CaM, we studied the effect of CaM antagonists on the CICR in skinned muscle fibres of the rabbit. Ca2+ release was measured by microfluorometry with Fura-2. 2. A CaM antagonist, trifluoperazine (TFP), potentiated the CICR in a dose-dependent manner (10-300 microM) at pCa 6, where a simple reversal of the CaM effect would be inhibition of the CICR. Furthermore, 100 microM TFP sensitized the CICR to Ca2+. A similar effect was produced by other CaM antagonists that were tested: chlorpromazine, W-7, mastoparan, and peptide fragment of CaM-binding residues of CaM-dependent protein kinase II. 3. The biphasic effect of CaM on the CICR was observed even in the presence of high concentrations of CaM antagonists or CaM-bindings peptides. 4. From these results we suggest that CaM has a unique mode of action on the CICR which is quite different from the effect of CaM on known enzymes.

Animals↗
PubMed2004

The N-terminal Ca2+-independent calmodulin-binding site on the inositol 1,4,5-trisphosphate receptor is responsible for calmodulin inhibition, even though this inhibition requires Ca2+.

Calmodulin (CaM) is a ubiquitous Ca(2+)-sensor protein that plays an important role in regulating a large number of Ca(2+) channels, including the inositol 1,4,5-trisphosphate receptor (IP(3)R). CaM binds to the IP(3)R at Ca(2+)-dependent as well as at Ca(2+)-independent interaction sites. In this study, we have investigated the Ca(2+)-independent CaM-binding site for its role in the regulation of the Ca(2+)-dependent bell-shaped activation curve of the IP(3)R. Suramin, a polysulfonated napthylurea, displaced CaM in both the presence and the absence of Ca(2+). Suramin competed with CaM for binding to different peptides representing the previously identified CaM-binding sites on IP(3)R1. By interacting with the N-terminal Ca(2+)-independent CaM-binding site, suramin mimicked the functional effect of CaM and induced an allosteric but competitive inhibition of IP(3) binding. Therefore, suramin also potently inhibited IP(3)-induced Ca(2+) release (IICR) from permeabilized cells predominantly expressing IP(3)R1 (L15 fibroblasts) or IP(3)R3 (Lvec fibroblasts), even though the IP(3)R3 does not contain Ca(2+)-dependent CaM-binding sites. Furthermore, we have found that CaM(1234), a CaM mutated in its four EF hands, inhibited IICR in a Ca(2+)-dependent way with the same potency as CaM. We conclude that CaM inhibits IICR via the N-terminal binding site. The inhibition requires Ca(2+) but CaM itself is not the Ca(2+) sensor for the inhibition of the IP(3)R.

Animals↗