The identification of calmodulin-binding sites on mitochondria in cultured 3T3 cells

We have uniformly labeled calmodulin with tetramethyl rhodamine isothiocyanate (CaM-RITC) and used the derivative as a molecular probe in order to identify available, unoccupied calmodulin-binding sites. In mildly fixed (3% formalin) cultured 3T3 cells, the biologically active CaM-RITC bound predominantly to mitochondria. Binding was markedly reduced in the presence of 1 mM EGTA. Stelazine, a phenothiozine which binds to calmodulin, prevented the interaction of CaM-RITC with mitochondrial sites. A 10 fold excess of unlabeled CaM competitively inhibited binding. Fluorescently labeled troponin C and parvalbumin did not bind to mitochondria on any other cellular organelle. Rhodamine (TMRITC) alone did not bind to 3T3 mitochondria. Similar results were obtained using ¹²⁵I-calmodulin binding to isolated rat liver mitochondria. When solubilized mitochondrial proteins were subjected to calmodulin-Sepharose affinity chromatography and eluted with 1 mM EGTA, there were two major polypeptides 120,000 and 67,000 daltons and at least three minor species (100,000, 60,000 and 40,000 daltons). The interaction required an active Ca²⁺-CaM complex and is specific for CaM. Double fluorescent staining with CaM-RITC and fluorescein-labeled antibodies to tubulin and DNAase I revealed a mitochondrial distribution pattern similar to that of microtubule arrays but unrelated to actin cabling. There was no evidence that CaM-RITC directly interacted with either microtubules or microfilaments.     

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca²⁺-bound CaM but not Ca²⁺-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.     

Spatial diffusivity and availability of intracellular calmodulin

Calmodulin (CaM) is the major pathway that transduces intracellular Ca²⁺ increases to the activation of a wide variety of downstream signaling enzymes. CaM and its target proteins form an integrated signaling network believed to be tuned spatially and temporally to control CaM's ability to appropriately pass signaling events downstream. Here, we report the spatial diffusivity and availability of CaM labeled with enhanced green fluorescent protein (eGFP)-CaM, at basal and elevated Ca²⁺, quantified by the novel fluorescent techniques of raster image scanning spectroscopy and number and brightness analysis. Our results show that in basal Ca²⁺ conditions cytoplasmic eGFP-CaM diffuses at a rate of 10 μm²/s, twofold slower than the noninteracting tracer, eGFP, indicating that a significant fraction of CaM is diffusing bound to other partners. The diffusion rate of eGFP-CaM is reduced to 7 μm²/s when a large (646 kDa) target protein Ca²⁺/CaM-dependent protein kinase II is coexpressed in the cells. In addition, the presence of Ca²⁺/calmodulin-dependent protein kinase II, which can bind up to 12 CaM molecules per holoenzyme, increases the stoichiometry of binding to an average of 3 CaMs per diffusive molecule. Elevating intracellular Ca²⁺ did not have a major impact on the diffusion of CaM complexes. These results present us with a model whereby CaM is spatially modulated by target proteins and support the hypothesis that CaM availability is a limiting factor in the network of CaM-signaling enzymes.     

The subcellular localization of an unusual rice calmodulin isoform,OsCaM61, depends on its prenylation status

Calmodulin (CaM) is a small Ca²⁺-binding protein highly conserved in eukaryotes. We have reported previously a novel rice CaM-like protein (OsCaM61) which contains an N-terminal CaM domain and a C-terminal extension with a potential prenylation site. Here we report in vitro activity assays confirm OsCaM61 as a functional CaM. Using the green fluorescent protein (GFP) as a visual marker, we further studied the subcellular localization of OsCaM61 in stably transformed tobacco cells. The GFP-OsCaM61 fusion protein was membrane-associated whereas OsCaM61-GFP was mainly detected in the nucleoplasm. GFP-OsCaM61 was transported into the nucleoplasm upon a block in isoprenoid biosynthesis by mevinolin treatment of cells. These results indicate that the prenylated OsCaM61 molecules are mainly membrane-associated whereas its unprenylated counterparts are transported into the nucleoplasm. Thus, OsCaM61 may play functions in co-ordinating Ca²⁺ signaling with isoprenoid metabolism.     

Ultracytochemistry of calmodulin binding sites in myocardial cells by staining of frozen thin sections with colloidal gold-labeled calmodulin

Calmodulin (CaM) has been implicated as a multifunctional regulator of Ca2+ in the cytoplasm of cells. We have recently introduced biologically active colloidal gold-labeled CaM as a marker for identifying potential CaM binding sites (unoccupied by endogenous CaM at the time of fixation) by electron microscopy and have stained frozen thin sections of rat cardiac muscle with this conjugate. In the presence of Ca2+, gold particles indicating CaM binding sites were found localized on the sarcoplasmic reticulum, mitochondria, and gap junctions. Control tissue sections treated with EGTA or exposed to excess amounts of unlabeled native CaM before staining showed no binding. We believe that cytochemistry of potential CaM binding sites revealed by staining with labeled exogenous CaM is useful in correlating known biochemical reactions of CaM with particular cell activities.     

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four “EF hand” type Ca²⁺ binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca²⁺ ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca_n (n = 0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Calmodulin pharmacology

Calmodulin (CaM) is a major intracellular receptor for Ca²⁺. CaM is thus a crucial receptor to consider in pharmacological modification of cellular activity. Potential mechanisms by which drugs may modify CaM effectiveness are considered in the context of its interaction with Ca²⁺ and in turn with its various effectors. Some examples of established drug mechanisms are considered. A wide range of chemical compounds representing diverse pharmacological classes are anti-CaM under some conditions. No simple relationships have been established between molecular level events and therapeutic applicability of anti-CaM compounds.     

The many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

The impact of calmodulin on the cell cycle analyzed in a novel human cellular genetic system

Calmodulin (CaM) is the principle mediator of the Ca²⁺ signal in all eukaryotic cells. A huge variety of basic cellular processes including cell cycle control, proliferation, secretion and motility, among many others are governed by CaM, which regulates activities of myriads of target proteins. Mammalian CaM is encoded by three genes localized on different chromosomes all producing an identical protein. In this study, we have generated HeLa human cancer cells conditionally expressing CaM in a genetic background with all three genes inactivated by CRISPR/Cas9. We demonstrate that downregulation of ectopically expressed CaM is achieved after 120 h, when cells are arrested in the M phase of the cell cycle. We show for the first time that CaM downregulation in human cancer cells is followed by a multinucleated senescent state as indicated by expression of β-galactosidase as well as cell morphology typical for senescent cells. Our newly generated genetic system may be useful for the analysis of other CaM regulated processes in eukaryotic cells in the absence of endogenous CaM genes.     

1H, 15N and 13C resonance assignments of yeast Saccharomyces cerevisiae calmodulin in the Ca2+-free state

Calmodulin (CaM) is a small Ca²⁺-binding protein, which has been found in all of eucaryotic cells examined. CaMs isolated from various species have highly conserved amino acid sequence (more than 90% identical), and show the same biological functions. CaM isolated from the baker's yeast (Saccharomyces cerevisiae) (yCaM), however, shares only 60% identity in the amino acid sequence with CaM from vertebrate, and shows quite distinct conformational and biochemical properties compared with those of CaM from other species. The conformational details of yCaM, however, have not been revealed yet. We achieved the chemical shift assignments of yCaM (146 amino acids) in the apo-state using uniformly ¹⁵N- and ¹³C-labeled protein. Consequently, the resonances of 95% atoms in the backbone amides were successfully assigned.     

Dissecting cooperative calmodulin binding to CaM kinase II: a detailed stochastic model

Calmodulin (CaM) is a major Ca²⁺ binding protein involved in two opposing processes of synaptic plasticity of CA1 pyramidal neurons: long-term potentiation (LTP) and depression (LTD). The N- and C-terminal lobes of CaM bind to its target separately but cooperatively and introduce complex dynamics that cannot be well understood by experimental measurement. Using a detailed stochastic model constructed upon experimental data, we have studied the interaction between CaM and Ca²⁺-CaM-dependent protein kinase II (CaMKII), a key enzyme underlying LTP. The model suggests that the accelerated binding of one lobe of CaM to CaMKII, when the opposing lobe is already bound to CaMKII, is a critical determinant of the cooperative interaction between Ca²⁺, CaM, and CaMKII. The model indicates that the target-bound Ca²⁺ free N-lobe has an extended lifetime and may regulate the Ca²⁺ response of CaMKII during LTP induction. The model also reveals multiple kinetic pathways which have not been previously predicted for CaM-dissociation from CaMKII.     

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting

Calmodulin (CaM) is a highly conserved calcium (Ca²⁺) binding protein that transduces Ca²⁺ signals into downstream effects influencing a range of cellular processes, including Ca²⁺ homeostasis. The present study explores CaM expression when Ca²⁺ homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca²⁺ ATPase (PMCA) known to regulate intracellular Ca²⁺ through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca²⁺ flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.     

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca²⁺, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca²⁺, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca²⁺, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca²⁺ stabilized the closed conformation by a factor of two. We conclude that the Ca²⁺-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca²⁺-dependent structural dynamics of bound CaM.     

A Novel Trans Conformation of Ligand-Free Calmodulin

Calmodulin (CaM) is a highly conserved eukaryotic protein that binds specifically to more than 100 target proteins in response to calcium (Ca²⁺) signal. CaM adopts a considerable degree of structural plasticity to accomplish this physiological role; however, the nature and extent of this plasticity remain to be fully understood. Here, we report the crystal structure of a novel trans conformation of ligand-free CaM where the relative disposition of two lobes of CaM is different, a conformation to-date not reported. While no major structural changes were observed in the independent N- and C-lobes as compared with previously reported structures of Ca²⁺/CaM, the central helix was tilted by ∼90° at Arg75. This is the first crystal structure of CaM to show a drastic conformational change in the central helix, and reveals one of several possible conformations of CaM to engage with its binding partner.     

Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca^2 +-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca^2 + binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.     

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

Distinguishing unfolding and functional conformational transitions of calmodulin using ultraviolet resonance Raman spectroscopy

Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium‐binding protein exhibits a broad range of structural transitions, including domain opening and folding–unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well‐known, it has been challenging to examine the dynamics of transition between these structures in real‐time, due to a lack of suitable spectroscopic probes of CaM structure. In this article, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C‐terminal domain in response to Ca²⁺ binding. This spectral difference is entirely due to differences in tertiary contacts at the interdomain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation‐sensitive tyrosine residues, opening a path to time‐resolved studies of CaM dynamics using Raman spectroscopy.     

CFP荧光蛋白文库构建及FRET技术筛选钙调素结合蛋白

钙调素（Calmodulin,CaM）是细胞内Ca^2＋信号的主要受体,能够与靶蛋白相互结合调节靶蛋白的活性,在细胞增殖、分化、凋亡、迁移等过程中都起着重要作用。荧光共振能量转移（fluorescence resonance energy transfer,FRET）技术是目前研究蛋白质相互作用比较成熟的方法之一。作者通过Cre-loxP位点特异性重组技术构建了带有CFP荧光蛋白标记的文库,与YFP—CaM共同转染HEK293细胞,应用荧光共振能量转移技术（FRET）进行检测,挑取发生FRET作用的单个细胞,并进行单细胞PcR检测。由此扩增出的片段通过测序和蛋白序列数据库NCBI进行序列比对后,筛选出与CaM产生相互作用的蛋白。目前,已经通过这种方法成功地筛选到了一些与CaM相结合的蛋白,从而为进一步研究CaM蛋白在生理环境下的作用提供有利条件。     

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca²⁺. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using ¹⁵N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca²⁺ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca²⁺ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca²⁺.     

The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca²⁺ and multiple, direct associations of the Ca²⁺ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca²⁺ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca²⁺/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca²⁺/CaM and reveal that the Ca²⁺/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca²⁺/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca²⁺ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.