Calmodulin Isoforms Research Articles

Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Molecular Insights into the MLCK Activation by CaM.

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca2+) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally, we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

Non-Canonical Interaction between Calmodulin and Calcineurin Contributes to the Differential Regulation of Plant-Derived Calmodulins on Calcineurin.

Calmodulin (CaM) serves as an important Ca2+ signaling hub that regulates many protein signaling pathways. Recently, it was demonstrated that plant CaM homologues can regulate mammalian targets, often in a manner that opposes the impact of the mammalian CaM (mCaM). However, the molecular basis of how CaM homologue mutations differentially impact target activation is unclear. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isoforms, sCaM-1 and sCaM-4, share >90 and ∼78% identity with the mCaM, respectively, and activate CaN with comparable or reduced activity relative to mCaM. We used molecular dynamics (MD) simulations and fluorometric assays of CaN-dependent dephosphorylation of MUF-P to probe whether calcium and protein-protein binding interactions are altered by plant CaMs relative to mCaM as a basis for differential CaN regulation. In the presence of CaN, we found that the two sCaMs' Ca2+ binding properties, such as their predicted coordination of Ca2+ and experimentally measured EC50 [Ca2+] values are comparable to mCaM. Furthermore, the binding of CaM to the CaM binding region (CaMBR) in CaN is comparable among the three CaMs, as evidenced by MD-predicted binding energies and experimentally measured EC50 [CaM] values. However, mCaM and sCaM-1 exhibited binding with a secondary region of CaN's regulatory domain that is weakened for sCaM-4. We speculate that this secondary interaction affects the turnover rate (kcat) of CaN based on our modeling of enzyme activity, which is consistent with our experimental data. Together, our data describe how plant-derived CaM variants alter CaN activity through enlisting interactions other than those directly influencing Ca2+ binding and canonical CaMBR binding, which may additionally play a role in the differential regulation of other mammalian targets.

Calmodulin is involved in the dual subcellular location of two chloroplast proteins

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

Arabidopsis cyclic nucleotide-gated channel 6 is negatively modulated by multiple calmodulin isoforms during heat shock.

An increased concentration of cytosolic Ca2+ is an early response of plant cells to heat shock. Arabidopsis cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca2+ influx and is activated by cAMP. However, it remains unclear how the Ca2+ conductivity of CNGC6 is negatively regulated under the elevated cytosolic Ca2+ concentration. In this study, Arabidopsis calmodulin isoforms CaM1/4, CaM2/3/5, CaM6, and CaM7 were found to bind to CNGC6 to varying degrees, and this binding was dependent on the presence of Ca2+ and IQ6, an atypical isoleucine-glutamine motif in CNGC6. Knockout of CaM2, CaM3, CaM5, and CaM7 genes led to a marked increase in plasma membrane inward Ca2+ current under heat shock conditions; however, knockout of CaM1, CaM4, and CaM6 genes had no significant effect on plasma membrane Ca2+ current. Moreover, the deletion of IQ6 from CNGC6 led to a marked increase in plasma membrane Ca2+ current under heat shock conditions. Taken together, the data suggest that CNGC6-mediated Ca2+ influx is likely to be negatively regulated by CaM2/3/5 and CaM7 isoforms under heat shock conditions, and that IQ6 plays an important role in CaM binding and the feedback regulation of the channel.

The Phytophthora RXLR Effector Avrblb2 Modulates Plant Immunity by Interfering With Ca2+ Signaling Pathway.

In plants, subcellular fluctuations in Ca2+ ion concentration are among the earliest responses to biotic and abiotic stresses. Calmodulin, which is a ubiquitous Ca2+ ion sensor in eukaryotes, plays a major role in translating these Ca2+ signatures to cellular responses by interacting with numerous proteins located in plasma membranes, cytoplasm, organelles and nuclei. In this report, we show that one of the Phytophthora RXLR effector, Avrblb2, interacts with calmodulin at the plasma membrane of the plant cells. Using deletion and single amino acid mutagenesis, we found that calmodulin binds to the effector domain of Avrblb2. In addition, we show that most known homologs of Avrblb2 in three different Phytophthora species interact with different isoforms of calmodulin. Type of amino acids at position 69 in Avrblb2, which determines Rbi-blb2 resistance protein-mediated defense responses, is not involved in the Avrblb2-calmodulin interaction. Using in planta functional analyses, we show that calmodulin binding to Avrblb2 is required for its recognition by Rpi-blb2 to incite hypersensitive response. These findings suggest that Avrblb2 by interacting with calmodulin interfere with plant defense associated Ca2+ signaling in plants.

Calmodulin 2 Functions as an RNA Chaperone in Prokaryotic Cells

Plants express many calmodulin (CaM) isoforms. These proteins regulate the growth, development and environmental stress responses of plants by modulating targets. Herein, the Arabidopsis CaM2 isoform was found to be crucial for cold adaptation in prokaryotic cells, similar to the Escherichia coli cold shock protein CspA. Expressing CaM2 or CspA in the cold-sensitive E. coli BX04 mutant complemented the cold-sensitive phenotype under cold stress, but expression of CaM1, CaM7 or CML8 (CaM8) did not. Similar to RNA chaperones such as CspA, CaM2 strongly interacted with nucleic acids and its nucleic acid-binding capacity was much higher than that of CaM7, despite there being only a single amino acid difference between these two isoforms. Microscopic observation of CaM2-GFP revealed that CaM2 plays roles in both the nucleus and cytosol where RNA molecules are abundant. These results suggest that CaM2 can positively modulate cold stress responses by interacting with nucleic acid targets. Furthermore, CaM2 has both nucleic acid targets, similar to CaM7, and protein targets such as CAMTA3.

A retained intron in the 3'-UTR of Calm3 mRNA mediates its Staufen2- and activity-dependent localization to neuronal dendrites.

Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 (Calm3L ) mRNA Calm3L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3L mRNA to distal dendrites.

Divergent Soybean Calmodulins Respond Similarly to Calcium Transients: Insight into Differential Target Regulation.

Plants commonly respond to stressors by modulating the expression of a large family of calcium binding proteins including isoforms of the ubiquitous signaling protein calmodulin (CaM). The various plant CaM isoforms are thought to differentially regulate the activity of specific target proteins to modulate cellular stress responses. The mechanism(s) behind differential target activation by the plant CaMs is unknown. In this study, we used steady-state and stopped-flow fluorescence spectroscopy to investigate the strategy by which two soybean CaMs (sCaM1 and sCaM4) have evolved to differentially regulate NAD kinase (NADK), which is activated by sCaM1 but inhibited by sCaM4. Although the isolated proteins have different cation binding properties, in the presence of Mg2+ and the CaM binding domains from proteins that are differentially regulated, the two plant CaMs respond nearly identically to rapid and slow Ca2+ transients. Our data suggest that the plant CaMs have evolved to bind certain targets with comparable affinities, respond similarly to a particular Ca2+ signature, but achieve different structural states, only one of which can activate the enzyme. Understanding the basis for differential enzyme regulation by the plant CaMs is the first step to engineering a vertebrate CaM that will selectively alter the CaM signaling network.

Arabidopsis CaM1 and CaM4 Promote Nitric Oxide Production and Salt Resistance by Inhibiting S-Nitrosoglutathione Reductase via Direct Binding.

Salt is a major threat to plant growth and crop productivity. Calmodulin (CaM), the most important multifunctional Ca2+ sensor protein in plants, mediates reactions against environmental stresses through target proteins; however, direct proof of the participation of CaM in salt tolerance and its corresponding signaling pathway in vivo is lacking. In this study, we found that AtCaM1 and AtCaM4 produced salt-responsive CaM isoforms according to real-time reverse transcription-polymerase chain reaction analyses; this result was verified based on a phenotypic analysis of salt-treated loss-of-function mutant and transgenic plants. We also found that the level of nitric oxide (NO), an important salt-responsive signaling molecule, varied in response to salt treatment depending on AtCaM1 and AtCaM4 expression. GSNOR is considered as an important and widely utilized regulatory component of NO homeostasis in plant resistance protein signaling networks. In vivo and in vitro protein-protein interaction assays revealed direct binding between AtCaM4 and S-nitrosoglutathione reductase (GSNOR), leading to reduced GSNOR activity and an increased NO level. Overexpression of GSNOR intensified the salt sensitivity of cam4 mutant plants accompanied by a reduced internal NO level, whereas a gsnor deficiency increased the salt tolerance of cam4 plants accompanied by an increased internal NO level. Physiological experiments showed that CaM4-GSNOR, acting through NO, reestablished the ion balance to increase plant resistance to salt stress. Together, these data suggest that AtCaM1 and AtCaM4 serve as signals in plant salt resistance by promoting NO accumulation through the binding and inhibition of GSNOR. This could be a conserved defensive signaling pathway in plants and animals.

Aortic wall proteomic analysis in spontaneously hypertensive rats with a blood pressure decrease induced by 6-week load-free swimming.

Decreased arterial compliance is one of the earliest detectable manifestations of adverse structural and functional changes within the vessel wall in hypertension. The proteomic approach is a powerful technique to analyze a complex mixture of proteins in various settings. Physical activity level was negatively associated with blood pressure. Sixteen 4-week-old male spontaneously hypertensive rats (SHR) and 16 Wistar-Kyoto (WKY) rats were randomly divided into four groups: i) SHR exercise group, ii) SHR rest group, iii) WKY exercise group and iv) WKY rest group. In the SHR and WKY exercise groups, rats were treated with a 6-week load-free swimming protocol (1 h/day, 5 days/week). The blood pressure of the rats was tested by the CODATM2 single non-invasive blood pressure measurement appliance. After the 6-week swimming protocol, the total aorta excluding abdominal aorta was extracted. The proteins were separated by two-dimensional gel electrophoresis and identified via LC-mass spectrometry (MS)/MS. After 6-week load-free swimming, blood pressure decreased in the SHRs. Compared with sedentary SHRs, 11 spots on the 2D-gel showed a significant difference in exercised SHRs. Nine of these were chosen for further identification. There were 5 upregulated proteins (long-chain specific acyl-CoA dehydrogenase, heat shock protein β-1, isocitrate dehydrogenase subunit α, actin, α cardiac muscle 1 preprotein and calmodulin isoform 2) and 4 downregulated proteins (adipocyte-type fatty acid-binding protein, tubulin β-2C chain, 78 kDa glucose-regulated protein precursor and mimecan). Proteomics is an effective method to identify the target proteins of exercise intervention for hypertension.

Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin

Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K+ channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca2+). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies.

High-throughput transcriptome sequencing identifies candidate genetic modifiers of vulnerability to fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) is a leading cause of neurodevelopmental disability. Genetic factors can modify vulnerability to FASD, but these elements are poorly characterized. We performed high-throughput transcriptional profiling to identify gene candidates that could potentially modify vulnerability to ethanol's (EtOH's) neurotoxicity. We interrogated a unique genetic resource, neuroprogenitor cells from 2 closely related Gallus gallus lines having well-characterized robust or attenuated EtOH responses with respect to intracellular calcium mobilization and CaMKII/β-catenin-dependent apoptosis. Samples were not exposed to EtOH prior to analysis. We identified 363 differentially expressed genes in neuroprogenitors from these 2 lines. Kyoto Encyclopedia of Genes and Genomes analysis revealed several gene clusters having significantly differential enrichment in gene expression. The largest and most significant cluster comprised ribosomal proteins (38 genes, p = 1.85 × 10(-47) ). Other significantly enriched gene clusters included metabolism (25 genes, p = 0.0098), oxidative phosphorylation (18 genes, p = 1.10 × 10(-11) ), spliceosome (13 genes, p = 7.02 × 10(-8) ), and protein processing in the endoplasmic reticulum (9 genes, p = 0.0011). Inspection of gene ontogeny (GO) terms identified 24 genes involved in the calcium/β-catenin signals that mediate EtOH's neurotoxicity in this model, including β-catenin itself and both calmodulin isoforms. Four of the identified pathways with altered transcript abundance mediate the flow of cellular information from RNA to protein. Importantly, ribosome biogenesis also senses nucleolar stress and regulates p53-mediated apoptosis in neural crest. Human ribosomopathies produce craniofacial malformations and 11 known ribosomopathy genes were differentially expressed in this model of neural crest apoptosis. Rapid changes in ribosome expression are consistently observed in EtOH-treated mouse embryo neural folds, a model that is developmentally similar to ours. The recurring identification of ribosome biogenesis suggests it is a candidate modifier of EtOH vulnerability. These results highlight this approach's efficacy to formulate new, mechanistic hypotheses regarding EtOH's developmental damage.

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

The cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto- and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca(2+) /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).

Genotype-Phenotype Correlation in Induced Pluripotent Stem Cell (iPSC)Derived Cardiomyocytes Carrying Calmodulin Mutations

Background: De novo mutations in calmodulin genes have been recently associated with markedly prolonged QT interval and life-threatening ventricular arrhythmias in infants. Biochemical evidences suggested impaired calcium ion binding to mutant calmodulins. Calcium dependent inactivation (CDI) of L-type calcium channel (ICaL) involves the interaction between Ca2+-calmodulin and the channel protein. Aims: To explore the effects of two CALM1 mutations (F142L and D130G) on electrical activity and ICaL properties in human induced-pluripotent stem cells (iPSC)-derived cardiomyocytes (CM). Methods: Skin fibroblasts of two patients carrying the mutation CALM1-F142L or CALM1-D130G were reprogrammed to generate iPSC and these cells differentiated into CM; a healthy donor was selected as control. Two cell clones for each mutation were analyzed to rule out clone specificity. Patch clamp and micro electrode arrays (MEA) analyses were performed on isolated iPSC-derived CM and beating clusters at about 40 days of differentiation respectively. ICaL was isolated in the presence of calcium or barium ions as charge carriers. Results: CALM1-F142L iPSC-derived CM showed prolonged field potential duration (FPD) with high beta-adrenergic sensitivity; peak ICaL density was unaltered, but CDI was markedly reduced and inactivation incomplete. CALM1-D130G iPSC-derived CM showed higher ICaL density and unaltered CDI. Conclusions: F142L and D130G calmodulin mutations differently affect ICaL properties in human iPSC-derived CM. F142L mutation is consistent with reduced calcium affinity of calmodulin; D130G abnormality suggests ICaL facilitation instead, possibly resulting from upregulation of alternative calmodulin isoforms. Overall, both calmodulin mutations might potentially cause arrhythmogenic calcium overload.

Comparing the Calcium Binding Abilities of Two Soybean Calmodulins: Towards Understanding the Divergent Nature of Plant Calmodulins

The discovery that plants contain multiple calmodulin (CaM) isoforms of variable sequence identity to animal CaM suggested an additional level of sophistication in the intracellular role of calcium regulation in plants. Past research has focused on the ability of conserved or divergent plant CaM isoforms to activate both mammalian and plant protein targets. At present, however, not much is known about how these isoforms respond to the signal of an increased cytosolic calcium concentration. Here, using isothermal titration calorimetry and NMR spectroscopy, we investigated the calcium binding properties of a conserved (CaM1) and a divergent (CaM4) CaM isoform from soybean (Glycine max). Both isoforms bind calcium with a semisequential pathway that favors the calcium binding EF-hands of the C-terminal lobe over those of the N-terminal lobe. From the measured dissociation constants, CaM4 binds calcium with a threefold greater affinity than CaM1 (K(d,Ca,mean) of 5.0 versus 14.9 μM) but has a significantly reduced selectivity against the chemically similar magnesium cation that binds preferentially to EF-hand I of both isoforms. The implications of a potential magnesium/calcium competition on the activation of CaM1 and CaM4 are discussed in context with their ability to respond to stimulus-specific calcium signatures and their known physiological roles.

Characterization of the microtubule-binding activity of kinesin-like calmodulin binding protein from Dunaliella salina

Although the C-terminal motor and the N-terminal myosin-like domains of KCBP in Dunaliella salina (DsKCBP) are implicated in interaction with the microtubules, its microtubule binding property has not been addressed. It has been shown that several calmodulin isoforms suppress the microtubule binding activity of KCBP, but whether the calmodulin-like protein (CLP) has this ability remains unknown. The results of our previous study showed that there are two microtubule binding sites in DsKCBP, motor domain at the C-terminus and MyTH4-FREM at the N-terminus. In the present study, MyTH4, without the companion of FERM, was identified as the minimal domain responsible for interaction with the microtubules in the N-terminal of DsKCBP. CLP interacted with the calmodulin-binding domain of DsKCBP in the presence of Ca2+, and inhibited the microtubule-binding activity of motor domain but not MyTH4 domain. Furthermore, MyTH4 domain in the N-terminus of DsKCBP was responsible for binding to the microtubules, and had 10-fold weaker affinity to the microtubules than the motor domain.

Involvement of Ca2+ and CaM3 in Regulation of Thermotolerance in Lily (Lilium longiflorum)

Involvement of calcium (Ca2+) and different calmodulin (CaM) isoforms in heat shock (HS) signal transduction in lily (Lilium longiflorum) was investigated in this study. Application of CaCl2 enhanced the thermotolerance of lily, whereas treatment with calcium ion chelator EGTA and CaM antagonist trifluoperazine (TFP) lowered the thermotolerance. Besides, HS-induced expression of LlHsf1 and LlHsfA2 genes were up-regulated by CaCl2 and down-regulated by EGTA or TFP. These findings implied that Ca2+–CaM were involved in HS signal transduction in lily via HSF pathway. Subsequently, five LlCaM genes encoding three canonical CaM isoforms were isolated and characterized, among which the LlCaM3 expression was induced by heat and CaCl2 and correlated positively with thermotolerance of lily. Additionally, transient expression analysis determined LlCaM3 to be a cytoplasm and nucleus protein. Furthermore, overexpression of LlCaM3 in Arabidopsis (Arabidopsis thaliana) increased the transcript level of AtHsfA1a and AtHsp18.2 following HS and improved the thermotolerance of transgenic plants. Taken together, the results suggest that LlCaM3 is a key component in Ca2+–CaM HS signaling pathway in lily and may be in the upstream of HSF.

Calcium and Magnesium Binding Properties of Soybean Calmodulin 1 and 4

Calmodulin (CaM) is known to bind and regulate hundreds of different proteins in a calcium dependent manner. Mammals encode a single isoform of CaM, while plants encode up to ten different isoforms (and dozens of calmodulin-like proteins). Moreover, recent studies have demonstrated that certain soybean CaMs have the ability to differentially activate several mammalian target enzymes in a competitive manner. As a first step in understanding the functional differences among these CaMs, we have studied the Ca2+ and Mg2+ binding properties of two unique soybean CaMs, sCaM1 and sCaM4. CaM has both N-terminal and C-terminal pairs of Ca2+ binding domains (EF-hands). In order to follow cation binding and dissociation from these sCaMs we utilized various fluorescent probes (Trp, Tyr, bis-ANS and quin-2). For each sCaM, our analysis revealed that the rate of Ca2+ dissociation was similar to the regional structural change occurring at each domain, which in turn was similar to the rate of closure of each domain's hydrophobic pocket. Compared to sCaM4, sCaM1 displayed a ∼2-fold lower affinity for Ca2+ at each domain. Consistent with this finding, the N- and C-terminal domains of sCaM1 had ∼5- and 2-fold faster calcium dissociation rates than the respective domains of SCaM4. Both sCaM's N-terminal domain bound Mg2+ with a physiologically relevant affinity (≤1mM), whereas their C-terminal domains had non-physiological Mg2+ binding affinities (≥6mM). The binding of Mg2+ to the N-terminal domains of the SCaMs kept the domain in the closed state and drastically delayed the rate of Ca2+ association and opening of the hydrophobic pocket. Thus, in addition to their ability to differentially regulate enzymes, these soybean CaMs also sense and respond to Ca2+ differently.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts

The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.