Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.     

The distribution of calmodulin and Ca^2＋—activated calmodulin in cell cycle of mouse erythroleukemia cells

Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation.Prior data from synchronized cell population could not actually stand for various CaM levels in different phases of cell cycle.Here,based upon quantitative measurement of fluorescence in individual cells,a method was developed to investigate intracellular total CaM and Ca^2 -activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level,and Ca^2 -activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP).In mouse erythroleukemia (MEL) cells,total CaM level increased from G1 through S to G2M,reaching a maximum of 2-fold increase,then reduced to half amount after cell division.Meanwhile,Ca^2 -activated CaM also in creased through the cell cycle(G1,S,G2M).Increasing observed in G1 meant that the entry of cells from G1 into S phase may require CaM accumulation,and,equally or even more important,Ca^2 -dependent activation of CaM.Ca^2 -activated CaM decreased after cell division.The results suggested that CaM gene expression and C^2 -modulated CaM activation act synergistically to accomplish the cell cycle progression.     

Interaction of α-N-acetyl-β-endorphin and calmodulin

Acetylation at the α-amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide β-endorphin, α-N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for α-N-acetyl-β-endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like β-endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to β-endorphin, suggest that residues 14–24 exhibit α-helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the α-amino terminal of β-endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, β-endorphin and the α-N-acetylated peptide behave very similarly with respect to calmodulin association.     

Does aluminum inhibit pollen germination via extracellular calmodulin?

The effect of aluminum (Al) on pollen germination and its mechanism of action were investigated. Pollen germination and pollen tube elongation were inhibited by Al at pH 4.5. This inhibitory effect was reversed by the addition of purified calmodulin (CaM), whereas neither the calcium binding-protein S-100 nor Al chelator citric acid at the same concentrations had any obvious effect on Al-inhibited pollen germination. The presence of either the membrane-impermeable CaM inhibitor anti-CaM antiserum or Ca2+ chelator EGTA completely suppressed the effect of exogenous CaM. These results indicate the involvement of extracellular calmodulin in the short-term effects of Al on pollen germination and pollen tube elongation.     

Immunoelectron microscopic localization of calmodulin in corn root cells

Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.     

Extracellular calmodulin: A polypeptide signal in plants?

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intra-cellular Ca2+ signals. But in the last ten years, it was found that CaM also exists and acts extracel-lularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP3, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no interce     

Calcium-dependent Pyk2 activation: a role for calmodulin?

Pyk2 (proline-rich tyrosine kinase 2) and FAK (focal adhesion kinase) are highly related tyrosine kinases. One distinguishing feature is the differential regulation of the two enzymes in response to elevation of cytoplasmic calcium. In the latest issue of the Biochemical Journal, Sasaki and co-workers have provided insight into the calcium-dependent regulation of Pyk2. The findings suggest that calmodulin may bind the FERM (4.1/ezrin/radixin/moesin) domain to promote Pyk2 activation in response to calcium signals triggered by vasopressin. While the molecular details of the protein-protein interaction and mechanism of activation remain to be firmly established, this study is the first to provide mechanistic insight into the regulation of Pyk2 by calcium.     

Do calcium and calmodulin trigger maturation in amphibian oocytes?

In contrast to earlier reports (J. L. Maller and E. G. Krebs, 1980, Curr. Top. Cell. Regul. 16, 271-311; M. Moreau, J. P. Vilian, and P. Guerrier, 1980, Dev. Biol. 78, 201-214; W. J. Wasserman and L. D. Smith, 1981, J. Cell Biol. 89, 389-394; D. Huchon, R. Ozon, E. H. Fischer, and J. G. Demaille, 1981, Mol. Cell. Endocrinol. 22, 211-222) calmodulin preparations isolated from Xenopus laevis ovaries or obtained commercially rarely induced maturation upon microinjection into individual oocytes. Calmodulin injections did result in significant cases of maturation when oocytes were first pretreated (primed) with calcium-free (EGTA) OR-2 and then injected in regular OR-2 medium. However, under these conditions the injected buffer solution alone was sometimes found to induce maturation. Under more optimal priming conditions, cases were found where as high as 100% of the oocytes matured simply by returning them to regular OR-2 medium. To determine which divalent cations could be involved in the priming effects of EGTA pretreatment we repeated the earlier ionophore work of W. J. Wasserman and Y. Masui (1975, J. Exp. Zool. 193, 369-375), looking not just at calcium and magnesium but other divalent cations as well. Several divalent cations (10 mM) were found to induce germinal vesicle breakdown with the following tentative order of efficacy, Co2+ greater than or equal to Zn2+ greater than or equal to Mn2+ greater than Ca2+ greater than Mg2+ greater than Ba2+, regardless of whether or not ionophore A23187 was present. These results, along with other reports in the literature, are discussed with respect to the theory that a rise in free calcium and calmodulin is involved in triggering oocyte maturation; we conclude that neither is involved.     

Conservation of the 3′-untranslated regions of calmodulin mRNAs in cetaceans

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5- and 3-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3- untranslated region (3UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3UTR sequences. Such high conservation of the 3UTRs suggests a specialized and significant function for this region in mammals.     

Distribution and role of calmodulin in tip growing hyphae of Saprolegnia ferax

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Molecular dynamics studies on conformational thermodynamics of Orai1–calmodulin complex

Molecular understanding of bio-macromolecular binding is a challenging task due to large sizes of the molecules and presence of variety of interactions. Here, we study the molecular mechanism of calmodulin (CaM) binding to Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. Although experimental observations indicate that Orai1 binds to the C-terminal of Ca²⁺-loaded CaM, it is not decisive if N-domain of CaM interacts with Orai1. We address the issue of interaction of different domains of CaM with Orai1 using conformational thermodynamic changes, computed from histograms of dihedral angles over simulated trajectories of CaM, CaM-binding domain of Orai1 and complexes of CaM with Orai1. The changes for all residues of both C and N terminal domains of CaM upon Orai1 binding are compared. Our analysis shows that Orai1binds to both C-terminal and N-terminal domains of CaM, indicating 1:2 stoichiometry. The Orai1 binding to N-terminal domain of CaM is less stable than that to the C-terminal domain. The binding residues are primarily hydrophobic. These observations are in qualitative agreement to the experiments. The conformational thermodynamic changes thus provide a useful computational tool to provide atomic details of interactions in bio-macromolecular binding.     

α-Calcium calmodulin kinase II modulates the temporal structure of hippocampal bursting patterns

The alpha calcium calmodulin kinase II (α-CaMKII) is known to play a key role in CA1/CA3 synaptic plasticity, hippocampal place cell stability and spatial learning. Additionally, there is evidence from hippocampal electrophysiological slice studies that this kinase has a role in regulating ion channels that control neuronal excitability. Here, we report in vivo single unit studies, with α-CaMKII mutant mice, in which threonine 305 was replaced with an aspartate (α-CaMKII(T305D) mutants), that indicate that this kinase modulates spike patterns in hippocampal pyramidal neurons. Previous studies showed that α-CaMKII(T305D) mutants have abnormalities in both hippocampal LTP and hippocampal-dependent learning. We found that besides decreased place cell stability, which could be caused by their LTP impairments, the hippocampal CA1 spike patterns of α-CaMKII(T305D) mutants were profoundly abnormal. Although overall firing rate, and overall burst frequency were not significantly altered in these mutants, inter-burst intervals, mean number of intra-burst spikes, ratio of intra-burst spikes to total spikes, and mean intra-burst intervals were significantly altered. In particular, the intra burst intervals of place cells in α-CaMKII(T305D) mutants showed higher variability than controls. These results provide in vivo evidence that besides its well-known function in synaptic plasticity, α-CaMKII, and in particular its inhibitory phosphorylation at threonine 305, also have a role in shaping the temporal structure of hippocampal burst patterns. These results suggest that some of the molecular processes involved in acquiring information may also shape the patterns used to encode this information.     

Do calmodulin binding IQ motifs have built‐in capping domains?

Most calmodulin (CaM) targets are α‐helices. It is not clear if CaM induces the adoption of an α‐helix configuration to its targets or if those targets are selected as they spontaneously adopt an α‐helical conformation. Other than an α‐helix propensity, there is a great variety of CaM targets with little more in common. One exception to this rule is the IQ site that can be recognized in a number of targets, such as those ion channels belonging to the KCNQ family. Although there is negligible sequence similarity between the IQ motif and the docking site on SK2 channels, both adopt a similar three‐dimensional disposition. The isolated SK2 target presents a pre‐folded core region that becomes fully α‐helical upon binding to CaM. The existence of this pre‐folded state suggests the occurrence of capping within CaM targets. In this review, we examine the capping properties within the residues flanking this core domain, and relate known IQ motifs and capping.     

Synergistic activation of bovine cerebellum adenylate cyclase by calmodulin and β-adrenergic agonists

The relationship between calmodulin-dependent and β-adrenergic-sensitive adenylate cyclase activities was examined in membrane preparations from bovine cerebellum. Although stimulation by β-adrenergic agonists or calmodulin can occur independently, it is shown that their simultaneous presence has a strong synergistic effect on enzyme activity. Calmodulin did not influence the regulatory components of the neurotransmitter-dependent pathway as shown by the lack of effect on (1) receptor affinity, (2) GTP requirement for receptor-mediated activation, (3) rate of activation by guanyl 5′-yl imidodiphosphate [Gpp(NH)p]. Conversely, isoproterenol and guanine nucleotides did not modify to a significant extent the characteristics of enzyme stimulation by Ca²⁺ and calmodulin. Furthermore, calmodulin and Gpp(NH)p-dependent activities displayed different sensitivities to thermal inactivation.Our results indicate that β-adrenergic agonists and calmodulin interact with the same catalytic activity in cerebellar membranes, but presumably via two independent pathways.     

Gap junction structure and cell-to-cell coupling regulation: is there a calmodulin involvement?

In most tissues neighboring cells communicate directly with each other by exchanging ions and small metabolites via cell-to-cell channels located at the intermembrane particles of gap junctions. Evidence indicates that the channels close when the [Ca2+]i or [H+]i increases. The channel occlusion (cell-to-cell uncoupling) is mainly a safety device by which cells can isolate themselves from damaged neighboring cells ("healing-over" process). Despite our knowledge of uncoupling agents, the uncoupling mechanism is still poorly understood. Uncoupling treatments have been shown to cause structural changes in gap junctions, characterized by an increase in tightness and regularity (crystallization) of particle packing and a decrease in particle size. Recently these changes have been shown to be induced by Ca2+ or H+ in isolated lens junctions and by Ca2+ in liver junctions, which suggests a close relationship between structural changes and uncoupling, but preliminary studies indicate that the junctional changes may not be synchronous with uncoupling but may lag behind it. However, recent X-ray diffraction data show that the channels of crystalline gap junctions (typical of uncoupled cells) are indeed closed, because they are inaccessible to sucrose (a gap junction permeant). Thus it seems that crystalline junctions are indeed in a non-permeable state, but the occlusion of the channels may precede the crystallization process. In the lens, junction crystallization is inhibited by a calmodulin (CaM) inhibitor, trifluoperazine (TFP). Is CaM involved in the uncoupling mechanism? To test this hypothesis, TFP and calmidazolium (CDZ), the most specific CaM inhibitor, were used on amphibian embryonic cells electrically uncoupled by CO2. Both TFP and CDZ effectively protect the cells from uncoupling, which suggests that CaM participates in the process. As a hypothesis, we propose that channel occlusion follows a CaM-mediated conformational change in the junctional protein. Particle crystallization may follow the conformational changes and result from a modification in electrostatic repulsion among the particles.     

Inhibition of human ether à go-go potassium channels by Ca(2+)/calmodulin

Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in XENOPUS: oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca(2+). Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca(2+)-activated potassium channels, Ca(2+) dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.     

Engineering of a novel Ca²⁺-regulated kinesin molecular motor using a calmodulin dimer linker

The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have "on-off" control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca(2+)-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca(2+)-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.     

Methylation of calmodulin at carboxylic acid residues in erythrocytes. A non-regulatory covalent modification?

The physiological role of protein carboxy-group methylation reactions in human erythrocytes was studied with calmodulin as an endogenous methyl-group acceptor. The steady-state degree of calmodulin carboxy-group methylation is substoichiometric both in intact cells and in a lysed-cell system (about 0.0003 mol of methyl groups/mol of polypeptide). Purified erythrocyte calmodulin is a substrate for a partially purified erythrocyte carboxy-group methyltransferase and can be methylated to the extent of about 0.0007-0.001 mol of methyl groups/mol of polypeptide. This erythrocyte protein methyltransferase displays an apparent specificity for atypical racemized and/or isomerized D-aspartate and L-isoaspartate residues [McFadden & Clarke (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464; Murray & Clarke (1984) J. Biol. Chem. 259, 10722-10732]. Exposure of calmodulin to elevated temperatures before methylation results in racemization of aspartate and/or asparagine residues, and may result in isoaspartate formation as well. The methylatability of these samples also increases as a function of time of heating, independent of the pH (over the range pH 5-9) or Ca2+ concentration; the most significant increase occurs during the initial 60 min, when calmodulin retains a fraction of its biological activity. These results are consistent with the hypothesis that methylation of calmodulin may occur at these uncommon aspartate residues, but are not consistent with a regulatory role for the methylation reaction.