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Changes in intracellular calcium (Ca2+) signaling can modulate cellular machinery required for cancer progression. Neuronal calcium sensor 1 (NCS1) is a ubiquitously expressed Ca2+‐binding protein that promotes tumor aggressiveness by enhancing cell survival and metastasis. However, the underlying mechanism by which NCS1 contributes to increased tumor aggressiveness has yet to be identified. In this study, we aimed to determine (a) whether NCS1 expression changes in response to external stimuli, (b) the importance of NCS1 for cell survival and migration, and (c) the cellular mechanism(s) through which NSC1 modulates these outcomes. We found that NCS1 abundance increases under conditions of stress, most prominently after stimulation with the pro‐inflammatory cytokine tumor necrosis factor α, in a manner dependent on nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB). We found that NFκB signaling is activated in human breast cancer tissue, which was accompanied by an increase in NCS1 mRNA expression. Further exploration into the relevance of NCS1 in breast cancer progression showed that knockout of NCS1 (NCS1 KO) caused decreased cell survival and motility, increased baseline intracellular Ca2+ levels, and decreased inositol 1,4,5‐trisphosphate‐mediated Ca2+ responses. Protein kinase B (Akt) activity was decreased in NCS1 KO cells, which could be rescued by buffering intracellular Ca2+. Conversely, Akt activity was increased in cells overexpressing NCS1 (NCS1 OE). We therefore conclude that NCS1 acts as cellular stress response protein up‐regulated by stress‐induced NFκB signaling and that NCS1 influences cell survival and motility through effects on Ca2+ signaling and Akt pathway activation., This study investigates the underlying mechanism by which the calcium (Ca2+)‐binding protein NCS1 promotes increased tumor aggressiveness. We characterize NCS1 as a stress response protein that is up‐regulated by exogenous stressors, including the cytokine tumor necrosis factor α, through a nuclear factor kappa‐light‐chain‐enhancer of activated B cells‐dependent mechanism. Increased NCS1 expression leads to disrupted intracellular Ca2+ signaling and activation of the protein kinase B pathway, leading to increased cell survival and migration.

A defection of blood circulation in the brain leads to ischemia, damage, and the death of nerve cells. It is known that individual populations of GABAergic neurons are the least resistant to the damaging factors of ischemia and therefore they die first of all, which leads to impaired inhibition in neuronal networks. To date, the neuroprotective properties of a number of calcium-binding proteins (calbindin, calretinin, and parvalbumin), which are markers of GABAergic neurons, are known. Neuronal calcium sensor-1 (NCS-1) is a signaling protein that is expressed in all types of neurons and is involved in the regulation of neurotransmission. The role of NCS-1 in the protection of neurons and especially their individual populations from ischemia and hyperexcitation has not been practically studied. In this work, using the methods of fluorescence microscopy, vitality tests, immunocytochemistry, and PCR analysis, the molecular mechanisms of the protective action of NCS-1 in ischemia/reoxygenation and hyperammonemia were established. Since NCS-1 is most expressed in GABAergic neurons, the knockdown of this protein with siRNA led to the most pronounced consequences in GABAergic neurons. The knockdown of NCS-1 (NCS-1-KD) suppressed the basic expression of protective proteins without significantly reducing cell viability. However, ischemia-like conditions (oxygen-glucose deprivation, OGD) and subsequent 24-h reoxygenation led to a more massive activation of apoptosis and necrosis in neurons with NCS-1-KD, compared to control cells. The mass death of NCS-1-KD cells during OGD and hyperammonemia has been associated with the induction of a more pronounced network hyperexcitation symptom, especially in the population of GABAergic neurons, leading to a global increase in cytosolic calcium ([Ca2+]i). The symptom of hyperexcitation of neurons with NCS-1-KD correlated with a decrease in the level of expression of the calcium-binding protein-parvalbumin. This was accompanied by an increase in the expression of excitatory ionotropic glutamate receptors, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) against the background of suppression of the expression of glutamate decarboxylase (synthesis of γ-aminobutyric acid).

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10–30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

Calcium (Ca2+) signaling is critical for neuronal functioning and requires the concerted interplay of numerous Ca2+-binding proteins, including neuronal calcium sensor 1 (NCS1). Although an important role of NCS1 in neuronal processes and in neurodevelopmental and neurodegenerative diseases has been established, the underlying mechanisms remain enigmatic. Here, we systematically investigated the functions of NCS1 in the brain. Using Golgi-Cox staining, we observed a reduction in dendritic complexity and spine density in the prefrontal cortex and the dorsal hippocampus of Ncs1(−/−) mice, which may underlie concomitantly observed deficits in memory acquisition. Subsequent RNA sequencing of Ncs1(−/−) and Ncs1(+/+) mouse brain tissues revealed that NCS1 modulates gene expression related to neuronal morphology and development. Investigation of developmental databases further supported a molecular role of NCS1 during brain development by identifying temporal gene expression patterns. Collectively, this study provides insights into NCS1-dependent signaling and lays the foundation for a better understanding of NCS1-associated diseases.

Neuronal calcium sensor 1 (NCS1), a calcium-binding protein, and transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, are fundamental in the regulation of calcium homeostasis. The interactions of these proteins and their regulation by paclitaxel (PTX) were investigated using biochemical, pharmacological, and electrophysiological approaches in both a breast cancer epithelial cell model and a neuronal model. TRPV4 and NCS1 reciprocally immunoprecipitated each other, suggesting that they make up a signaling complex. The functional consequence of this physical association was that TRPV4 currents increased with increased NCS1 expression. Calcium fluxes through TRPV4 correlated with the magnitude of TRPV4 currents, and these calcium fluxes depended on NCS1 expression levels. Exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes but decreased the expression of NCS1. These findings augment the understanding of the properties of TRPV4, the role of NCS1 in the regulation of TRPV4, and the cellular mechanisms of PTX-induced neuropathy. SIGNIFICANCE STATEMENT TRPV4 and NCS1 physically and functionally interact. Increased expression of NCS1 enhances TRPV4-dependent currents, which are further amplified by treatment with the chemotherapeutic drug paclitaxel, an effect associated with adverse effects of chemotherapy, including neuropathy.

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways.

Neuronal calcium sensor-1 (NCS-1) has been identified as a binding partner of the taxane, paclitaxel. Our previous study showed that overexpression of NCS-1 increased the efficacy of paclitaxel in vitro, but was associated with poor clinical outcome. Here, we determine if NCS-1 expression is associated with pathological complete response (pCR) to taxane-based neoadjuvant chemotherapy in 105 pre-treatment breast cancer biopsies. Elevated expression of NCS-1 was found to be positively associated with pCR. These results suggest that NCS-1 may be a predictive biomarker for response to taxane-based neoadjuvant chemotherapy in breast cancer.

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. The causes of PD in humans are still unknown, although metabolic characteristics of the neurons affected by the disease have been implicated in their selective susceptibility. Mitochondrial dysfunction and proteostatic stress are recognized to be important in the pathogenesis of both familial and sporadic PD, and they both culminate in bioenergetic deficits. Exposure to calcium overload has recently emerged as a key determinant, and pharmacological treatment that inhibits Ca2+ entry diminishes neuronal damage in chemical models of PD. In this review, we first introduce general concepts on neuronal Ca2+ signaling and then summarize the current knowledge on fundamental properties of substantia nigra pars compacta dopaminergic neurons, on the role of the interplay between Ca2+ and dopamine signaling in neuronal activity and susceptibility to cell death. We also discuss the possible involvement of a “neglected” player, the Neuronal Calcium Sensor-1 (NCS-1), which has been shown to participate to dopaminergic signaling by regulating dopamine dependent receptor desensitization in normal brain but, data supporting a direct role in PD pathogenesis are still missing. However, it is intriguing to speculate that the Ca2+-dependent modulation of NCS-1 activity could eventually counteract dopaminergic neurons degeneration.

We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca2+ stress in Neurospora crassa. Increasing concentration of Ca2+ increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca2+ concentrations. These results suggested the regulation of NCS-1 function through calcineurin- CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that the CRZ-1 binds specifically to an 8 bp sequence 5'-CCTTCACA-3' in the ncs-1 promoter 216 bp upstream of the ATG start codon. We also showed that NCS-1 binds to the Ca2+ permeable channel MID-1 for tolerance to Ca2+ stress. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter, causing upregulation of NCS-1 that binds to MID-1 for tolerance to Ca2+ stress.

International audience; Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca 2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn 2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn 2+ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 10 6 M −1) and one low-affinity (2 × 10 5 M −1) Zn 2+-binding sites, whereas Mg 2+-loaded and Ca 2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn 2+-binding sites of apo-and Mg 2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn 2+ concentrations displace Mg 2+ from the EF-hands and decrease the stoichiometry of Ca 2+ binding. Meanwhile, one of the EF-hands of Zn 2+-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn 2+ binding to Ca 2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca 2+-free NCS-1 and favours the interaction of its Ca 2+-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding Frontiers in Molecular Neuroscience | www.frontiersin.org 1

Neuronal Calcium Sensor-1 (NCS1) belongs to the family of Neuronal Calcium Sensor (NCS) proteins. NCS1 is composed of four EF-hand motifs and an N-terminal myristoylation. However, the presence of a calcium-myristoyl switch in NCS1 and its role in the membrane binding are controversial. The model of Langmuir lipid monolayers is thus used to mimic the cell membrane in order to characterize the membrane interactions of NCS1. Two binding parameters are calculated from monolayer measurements: the maximum insertion pressure, up to which protein binding is energetically favorable, and the synergy, reporting attractive or repulsive interactions with the lipid monolayers. Binding membrane measurements performed in the presence of myristoylated NCS1 reveal better binding interactions for phospholipids composed of phosphoethanolamine polar head groups and unsaturated fatty acyl chains. In the absence of calcium, the membrane binding measurements are drastically modified and suggest that the protein is more strongly bound to the membrane. Indeed, the binding of calcium by three EF-hand motifs of NCS1 leads to a conformation change. NCS1 arrangement at the membrane could thus be reshuffled for better interactions with its substrates. The N-terminal peptide of NCS1 is composed of two amphiphilic helices involved in the membrane interactions of NCS1. Moreover, the presence of the myristoyl group has a weak influence on the membrane binding of NCS1 suggesting the absence of a calcium-myristoyl switch mechanism in this protein. The myristoylation could thus have a structural role required in the folding/unfolding of NCS1 which is essential to its multiple biological functions.

Neuronal calcium sensor-1 or Frequenin is a calcium sensor widely expressed in the nervous system, with roles in neurotransmission, neurite outgrowth, synaptic plasticity, learning, and motivated behaviours. Neuronal calcium sensor-1 has been implicated in neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and bipolar disorder. However, the role of neuronal calcium sensor-1 in behavioural phenotypes and brain changes relevant to autism spectrum disorder have not been evaluated. We show that neuronal calcium sensor-1 deletion in the mouse leads to a mild deficit in social approach and impaired displaced object recognition without affecting social interactions, behavioural flexibility, spatial reference memory, anxiety-like behaviour, or sensorimotor gating. Morphologically, neuronal calcium sensor-1 deletion leads to increased dendritic arbour complexity in the frontal cortex. At the level of hippocampal synaptic plasticity, neuronal calcium sensor-1 deletion leads to a reduction in long-term potentiation in the dentate gyrus, but not area Cornu Ammonis 1. Metabotropic glutamate receptor-induced long-term depression was unaffected in both dentate and Cornu Ammonis 1. These studies identify roles for neuronal calcium sensor-1 in specific subregions of the brain including a phenotype relevant to neuropsychiatric disorders.

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca(2+) concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca(2+)-bound state, the presence of Mg(2+) allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kβ). Of a particular note, Ca(2+) induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca(2+)-binding to EF-1 is restored. Moreover, Ca(2+), which otherwise augments the enzymatic activity of PI4Kβ (modulated by NCS-1), leads to a further decline in the modulated PI4Kβ activity by myristoylated mutants (with canonical EF-1) pointing toward a loss of Ca(2+) signaling and specificity at the structural as well as functional levels. This study establishes the presence of the strong liaison between myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of Ca(2+) specific signal transduction to downstream effecter molecules despite Ca(2+) binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca(2+) sensitivity/specificity in NCS-1.

Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional Ca2+-binding protein that affects a range of cellular processes beyond those related to neurons. Functional characterization of NCS-1 in neuronal model systems suggests that NCS-1 may influence oncogenic processes. To this end, the biological role of NCS-1 was investigated by altering its endogenous expression in MCF-7 and MB-231 breast cancer cells. Overexpression of NCS-1 resulted in a more aggressive tumor phenotype demonstrated by a marked increase in invasion and motility, and a decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitaxel-induced cell death in a manner that was independent of cellular proliferation. To determine the association between NCS-1 and clinical outcome, NCS-1 expression was measured in two independent breast cancer cohorts by the Automated Quantitative Analysis method of quantitative immunofluorescence. Elevated levels of NCS-1 were significantly correlated with shorter survival rates. Furthermore, multivariate analysis demonstrated that NCS-1 status was prognostic, independent of estrogen receptor, progesterone receptor, HER2, and lymph node status. These findings indicate that NCS-1 plays a role in the aggressive behavior of a subset of breast cancers and has therapeutic or biomarker potential. Implications: NCS-1, a calcium-binding protein, is associated with clinicopathologic features of aggressiveness in breast cancer cells and worse outcome in two breast cancer patient cohorts. Mol Cancer Res; 15(7); 942–52. ©2017 AACR.

pH is highly regulated in the mammalian central nervous systems. Neuronal calcium sensor-1 (NCS-1) can interact with numerous target proteins. Compared with C. elegans, evolution has avoided histidine residues at positions 102 and 83 in the human and Xenopus laevis NCS-1 protein, possibly to decrease conformational sensitivity to pH gradients in synaptic processes. We used all-atom molecular dynamics (MD) simulations to investigate the effects of amino acid substitutions between species on human NCS-1 by substituting Arg102 and Ser83 for histidine (R102H and S83H) at neutral and acidic pH. Our cumulative 5 μs simulations revealed that the R102H mutation slightly increases the structural flexibility of loop L2 and the R102H(protonated) mutation decrease protein stability. Community network analysis illustrates that the R102H and S83H mutations weaken the inter-domain and strengthen the intra-domain correlations. Secondary structure contents in the S83H and the S83H(protonated) mutants are similar to those in the wild type (WT), whereas the global structural stabilities and salt-bridge probabilities decrease. This study highlights conformational dynamics effects of R102H and S83H mutations respectively on NCS-1’s local structural flexibility and global stability, while the protonated histidine decreases the stability. Thus, histidines at positions 102 and 83 may not be compatible with NCS-1’s function whether in neutral or protonated states.

Neuronal calcium sensor-1 (NCS-1) protein has a variety of different neuronal functions and interacts with multiple binding partners mostly through a large solvent-exposed hydrophobic crevice (HC). A single R102Q mutation in human NCS-1 protein was demonstrated to be associated with autism disease. Solution NMR study reported that this R102Q mutant had long-range chemical shift effects on the HC and the C-terminal tail (L3). To understand the influence of the R102Q mutation on the HC and L3 of NCS-1, we have investigated the conformational dynamics and the structural flexibility of wild type (WT) NCS-1 and its R102Q mutant by conducting extensive all-atom molecular dynamics (MD) simulations. On the basis of six independent 450 ns MD simulations, we have found that the R102Q mutation in NCS-1 protein (1) dramatically reduces the flexibility of loops L2 and L3, (2) facilitates L3 in a more extended state to occupy the hydrophobic crevice to a larger extent, (3) significantly affects the intersegment salt bridges, and (4) changes the subspace of the free energy landscape of NCS-1 protein. Analysis of the salt bridge network in both WT and the R102Q variant demonstrates that the R102Q-mutation-induced salt bridge alternations play a critical role on the reduced flexibility of L2 and L3. These results reveal the important role of salt bridges on the structural properties of NCS-1 protein and that R102Q mutation disables the dynamic relocation of C-terminus, which may block the binding of NCS-1 protein to its receptors. This study may provide structural insights into the autistic spectrum disorder associated with R102Q mutation.

The calcium (Ca2+) signalling system in the filamentous fungus Neurospora crassa is unique and significantly different from that in plants and animals, mainly with regard to the second messenger systems involved in Ca2+-release from internal stores (Galagan et al. 2003). The complex Ca2+ signalling system in N. crassa contains 48 Ca2+ signalling proteins including a Ca2+ and/or CaM binding protein called neuronal calcium sensor-1 (NCS-1), a Ca2+-permeable channel MID-1 and a PMCA-type Ca2+-ATPase NCA-2 (Zelter et al. 2004; Tamuli et al. 2013). Here, we show that ncs-1, mid-1 and nca-2 interactions affect growth, carotenoid accumulation, Ca2+ stress tolerance, ultraviolet (UV) survival and circadian-regulated conidiation in N. crassa. The N. crassa homologue of NCS-1 has a role in growth, Ca2+ stress tolerance and UV survival (Deka et al. 2011; Tamuli et al. 2011). MID-1 is necessary for Ca2+homeostasis in N. crassa (Lew et al. 2008). Mid-1 in Gibberella zeae has a role in growth, development and ascospore discharge (Cavinder et al. 2011). NCA-2 is responsible for adaptation to stress conditions and functions to pump Ca2+ out of the cell in N. crassa (Benito et al. 2000; Bowman et al. 2011). In Schizosaccharomyces pombe, Ncs1p, the homologue of NCS-1, promotes Ca2+-induced closure of Yam8p Ca2+ channel, a homologue of the MID1 (Hamasaki-Katagiri and Ames 2010). The Ca2+-sensitive phenotype of the ncs1 deletion mutant is rescued by a yam8 deletion, indicating that Ncs1p negatively regulates Yam8p in S. pombe

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

Neuronal calcium sensor-1 (NCS-1) protein has orthologues from Saccharomyces cerevisiae (Frq1) to human with highly conserved amino acid sequences. NCS-1 is an important factor controlling the animal’s response to temperature change. This leads us to investigate the temperature effects on the conformational dynamics of the human NCS-1 at 310 K and 316 K by extensive all-atom explicit-solvent molecular dynamics (MD) simulations and dynamic community network analysis. Four independent 500-ns MD simulations shows that the secondary structure content at 316 K is similar to that at 310 K, whereas the global protein structure is expanded at the elevated temperature. The elevated temperature results in loop 3 (L3) adopting an extended state that occupies the hydrophobic crevice, thereby blocking the binding of NCS-1 protein to its receptors. The number of suboptimal communication paths starting at residue D176 and ending at V190 in the L3 C-terminal tail is reduced with the elevation of temperature. The dynamic community network analysis suggests that the correlation between the N- and C-domain is weakened and the intradomain coupling is strengthened at 316 K. The elevation of temperature reduces the number of the salt bridges, especially in the C-domain, which may induce the conformational dynamic changes. This study suggests that the elevated temperature affects the conformational dynamics of human NCS-1 protein. Comparison of the structural dynamics of R102Q mutant and Δ176–190 truncated NCS-1 suggests that the structural and dynamics response of NCS-1 protein to elevation of temperature may be one of its intrinsic functional properties.

Neuronal calcium sensor proteins and their homologues participate in transducing extracellular signals that affect intracellular Ca(2+) levels, which in turn regulate enzyme activities, secretion, gene expression and other biological processes. The filamentous fungus Beauveria bassiana is a broad-host-range pathogen of insects that acidifies the extracellular milieu during growth and pathogenesis towards target hosts. A collection of B. bassiana random insertion mutants were screened on pH indicator plates and one mutant was isolated that displayed reduced acidification. The random insertion site was mapped to a gene that displayed homology to the neuronal calcium sensor/frequenin protein family and was designated Bbcsa1. To validate the role of Bbcsa1 in B. bassiana, a targeted gene-knockout was constructed. Data confirmed that Bbcsa1 was not an essential gene and the ΔBbcsa1 strain displayed delayed acidification of the medium when grown in Czapek-Dox medium, as compared with the wild-type parent. HPLC profiling of secreted metabolites did not detect any major changes in the production of organic acids, although downregulation of the membrane H(+) pump/ATPase was noted in the mutant. A slight growth-deficient phenotype was observed for the ΔBbcsa1 strain on Czapek-Dox and potato dextrose media, which was accentuated at high calcium concentrations (500 mM) and 1.5 M sorbitol, but was unaffected by EDTA or SDS. Perturbations in vacuole morphology were also noted for the mutant. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the ΔBbcsa1 mutant when applied topically, representing the natural route of infection, but no significant effect was observed when fungal cells were directly injected into target hosts. These results suggest that Bbcsa1 participates in pre-penetration or early penetration events, but is dispensable once the insect cuticle has been breached.

Calcium (Ca(2+)) is an important intracellular messenger, regulating myocyte contraction via excitation-contraction (EC) coupling and gene transcription underlying hypertrophy in the heart. Although the mechanisms of EC coupling in the immature heart are believed to be different from those in the adult heart because of the structural immaturity of the sarcoplasmic reticulum in the young heart, the details of these mechanisms are not completely understood. Neuronal Ca(2+) sensor-1 (NCS-1) is an EF-hand Ca(2+)-binding protein that is highly expressed in young hearts; however, little is known about its cardiac functions. In this review, we summarize our recent findings indicating that NCS-1 acts as a novel regulator enhancing Ca(2+) signals in the heart and hence promoting contraction in the immature heart and hypertrophy in the adult heart. Possible signal transduction pathways are also discussed.

NCU04379 gene encodes a conserved Ca(2+) and/or calmodulin binding protein that possesses a consensus signal for N-terminal myristoylation and four EF-hands, characteristics of Neuronal Calcium Sensor-1proteins. The NCU04379.2 knockout mutant shows slow growth rate, increased sensitivity to calcium and ultraviolet (UV) irradiation, and a wild-type fragment carrying NCU04379 gene complements the mutant. Therefore, NCU04379 gene has a role in growth, calcium stress tolerance, and UV survival. Crosses homozygous for ΔNCU04379.2 mutant strains were fully fertile; however, we found evidence for involvement of Ca(2+)/calmodulin-dependent protein kinase encoding genes NCU02283 and NCU09123 in sexual development.

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca2+-binding sites, and mediates the effects of cytosolic Ca2+. Minute changes in free intracellular Ca2+ are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca2+/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca2+/CaMKs comes mostly from studies of the mammalian enzymes. Thus far very few fungal homologues of NCS-1 and Ca2+/CaMKs have been characterized and little is known about their cellular roles. In this minireview, we describe the known sequences, interactions with target proteins and cellular roles of NCS-1 and Ca2+/CaMKs in fungi. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Paclitaxel (Taxol) is a microtubule-stabilizing compound that is used for cancer chemotherapy. However, Taxol administration is limited by serious side effects including cardiac arrhythmia, which cannot be explained by its microtubule-stabilizing effect. Recently, neuronal calcium sensor 1 (NCS-1), a calcium binding protein that modulates the inositol-1,4,5-trisphosphate receptor (InsP(3)R), was described as a binding partner of Taxol and as a substrate of calpain. We examined calcium signaling processes in cardiomyocytes after treatment with Taxol to investigate the basis of Taxol-induced cardiac arrhythmia. After treating isolated neonatal rat ventricular myocytes with a therapeutic concentration of Taxol for several hours live cell imaging experiments showed that the frequency of spontaneous calcium oscillations significantly increased. This effect was not mimicked by other tubulin-stabilizing agents. However, it was prevented by inhibiting the InsP(3)R. Taxol treated cells had increased expression of NCS-1, an effect also detectable after Taxol administration in vivo. Short hairpin RNA mediated knockdown of NCS-1 decreased InsP(3)R dependent intracellular calcium release, whereas Taxol treatment, that increased NCS-1 levels, increased InsP(3)R dependent calcium release. The effects of Taxol were ryanodine receptor independent. At the single channel level Taxol and NCS-1 mediated an increase in InsP(3)R activity. Calpain activity was not affected by Taxol in cardiomyocytes suggesting a calpain independent signaling pathway. In short, our study shows that Taxol impacts calcium signaling and calcium oscillations in cardiomyocytes through NCS-1 and the InsP(3)R.

Neuronal Calcium Sensor-1 (NCS-1) is a member of calcium sensor family. It is originally identified as frequenin. NCS-1 has been found to interact with membrane and cytosolic proteins and its physiological role is governed by N-terminal myristoylation. In this paper, we report the NMR assignments of both myristoylated and non-myristoylated NCS-1 in the presence of a membrane.

Neuronal calcium sensor (NCS) proteins regulate signal transduction and are highly conserved from yeast to humans. NCS homolog in fission yeast (Ncs1p) is essential for cell growth under extreme Ca(2+) conditions. Ncs1p expression increases approximately 100-fold when fission yeast grows in high extracellular Ca(2+) (0.1 M). Here, we show that Ca(2+)-induced expression of Ncs1p is controlled at the level of transcription. Transcriptional reporter assays show that ncs1 promoter activity increased 30-fold when extracellular Ca(2+) was raised to 0.1 M and was highly Ca(2+)-specific. Ca(2+)-dependent transcription of ncs1 is abolished by the calcineurin inhibitor (FK506) and by knocking out the calcineurin target, prz1. Thus, Ca(2+)-induced expression of Ncs1p is linked to the calcineurin/prz1 stress response. The Ca(2+)-responsive ncs1 promoter region consists of 130 nucleotides directly upstream from the start codon and contains tandem repeats of the sequence, 5'-caact-3', that binds to Prz1p. The Ca(2+)-sensitive ncs1Delta phenotype is rescued by a yam8 null mutation, suggesting a possible interaction between Ncs1p and the Ca(2+) channel, Yam8p. Ca(2+) uptake and Ncs1p binding to yeast membranes are both decreased in yam8Delta, suggesting Ca(2+)-induced binding of Ncs1p to Yam8p results in channel closure. We propose that Ncs1p promotes Ca(2+) tolerance in fission yeast, in part by cytosolic Ca(2+) buffering and perhaps by negatively regulating the Yam8p Ca(2+) channel.

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1). The pharmacological inhibition of InsP(3) receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP(3)R1, which may play an important role in the regulation of neurite outgrowth.

We have developed a protocol to produce large quantities of high purity myristoylated and non-myristoylated neuronal calcium sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium-dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6x His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100-200 microM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50mg of homogenous myristoylated NCS-1 is obtained from 1L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 DeltaEF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.

To evaluate whether the thermotaxis tracking model is suitable for assessing long-term memory (LTM) in the nematode Caenorhabditis elegans.Animals were trained at 20 degrees C overnight in presence of food. The percentage of animals performing isothermal tracking (IT) behavior was measured at different time intervals after the training.The percentage of animals performing IT behavior, the numbers of body bends inside and outside the training temperature, and the expression patterns of AFD and AIY neurons were similar to those in control animals at 36 and 48 h after training; whereas when extending to 60, 72, and 84 h, locomotory behavior defects were observed in the assayed animals, suggesting that this thermal tracking model is feasible for analyzing LTM at 36 and 48 h after training. Moreover, the percentage of animals performing IT behavior was reduced at 18, 36, and 48 h after training in neuronal calcium sensor-1 gene (nsc-1) mutant animals compared with that in wild-type N2 animals. In addition, exposure to plumbum (Pb) significantly repressed the LTM at 18, 36, and 48 h after training in both wild-type N2 and ncs-1 mutant animals.The thermotaxis tracking model is suitable for evaluating the LTM regulated by NCS-1, and can be employed for elucidating regulatory functions of specific genes or effects of stimuli on memory in C. elegans.

Neuronal calcium sensor-1 (NCS-1) protein has been implicated in multiple neuronal functions by binding partners mostly through a largely exposed hydrophobic crevice (HC). In the absence of a ligand, the C-terminal tail (loop L3, residues D176 to V190) binds directly to the HC pocket as a ligand mimetic, occupying the HC and regulating its conformational stability. A recent experimental study reported that L3 deletion resulted in global structure destabilization. However, the influence of C-terminal tail on the conformations of NCS-1 protein is unclear at the atomic level. In this study, we investigated the structural properties and the conformational dynamics of wild type NCS-1 and L3 truncation variant by extensive all-atom molecular dynamics (MD) simulations. Our cumulative 2 μs MD simulations demonstrated that L3 deletion increased the structural flexibility of the C-domain and the distant N-domain. The community network analysis illustrated that C-terminal tail truncation weakened the interdomain correlation. Moreover, our data showed that the variant significantly disrupted the salt bridges network and expanded simultaneously the global structure and HC. These conformational changes caused by C-terminal tail truncation may affect the regulation of target interactions. Our study provides atomic details of the conformational dynamics effects of the C-terminal tail on human wild type NCS-1.

Background: Neuronal calcium sensor-1 (NCS-1) is a Ca2+-binding protein that interacts with neuronal proteins. Results: NCS-1 binds the dopamine D2 receptor and its cognate kinase in different ways. Conclusion: The C terminus and EF3-EF4 linker of NCS-1 modulate its binding to these target proteins. Significance: The specificity and diversity of NCS family proteins are determined by the C-lobe-binding site conformation., Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.

The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. Here we propose a mechanism involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein–protein interaction between NCS-1 and TRPC5, and involvement of this protein complex in retardation of neurite outgrowth.

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca(2+) binding and Ca(2+)-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The (45)Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca(2+) more strongly (with three Ca(2+) binding sites) than the non-myristoylated one (with two Ca(2+) binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca(2+). Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca(2+) binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca(2+) also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg(2+)-induced changes in near-UV CD are different from Ca(2+)-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca(2+), which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca(2+) binding, which might be crucial for its physiological functions.

The neuronal calcium sensor (NCS) family of Ca(2+)-binding proteins regulates a number of different processes in neurons and photoreceptor cells. The first of these proteins to be characterized, recoverin, was shown to exhibit a Ca(2+)/myristoyl switch whereby its N-terminal myristoyl group is sequestered in the Ca(2+)-free form and is exposed on Ca(2+) binding to allow the protein to become membrane-associated. It has subsequently been shown that certain other family members also exhibit this mechanism in living cells. In contrast, NCS-1 does not show the Ca(2+)/myristoyl switch and is membrane-associated even at low Ca(2+) concentrations. We have used sequence comparison combined with information from structural analyses to attempt to identify candidate residues within the NCS proteins that determine whether or not the Ca(2+)/myristoyl switch operates in cells and have tested their functional significance by mutagenesis. The results show that NCS-1 possesses residues within its N terminus that lock the myristoyl group in an exposed conformation. In addition, other structural aspects within the C-terminal domains are required to allow the switch to operate. We have determined a key role for residues within the motif EELTRK in NCS-1 in keeping the myristoyl group exposed and allowing the protein to be constitutively membrane-associated.

Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic α-helix and that frequenins bind to one side of this α-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.

Arabidopsis thaliana calcineurin B-like protein (AtCBL2) is a member of a recently identified family of calcineurin B-like calcium-binding proteins in A. thaliana. The crystal structure of AtCBL2 has been determined at 2.1 A resolution. The protein forms a compact alpha-helical structure with two pairs of EF-hand motifs. The structure is similar in overall folding topology to the structures of calcineurin B and neuronal calcium sensor 1, but differs significantly in local conformation. The two calcium ions are coordinated in the first and fourth EF-hand motifs, whereas the second and third EF-hand motifs are maintained in the open form by internal hydrogen bonding without coordination of calcium ions. Both a possible site and a possible mechanism for the target binding to AtCBL2 are discussed based on the three-dimensional structure.

Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.

The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca(2+), hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin delta, also translocated to this region after a rise in Ca(2+) concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin delta in the absence of calcium, but in the presence of 10 microm Ca(2+), both proteins translocated to and were retained in the perinuclear region. NCS-1 localization was unchanged in permeabilized cells regardless of calcium concentration. The localization of NCS-1 was unaffected by mutations in all functional EF hands, indicating that its localization was independent of Ca(2+). A minimal myristoylation motif (hippocalcin-(1-14)) fused to EGFP resulted in similar perinuclear targeting, showing that localization of these proteins is because of the exposure of the myristoyl group. This was confirmed by mutation of the myristoyl motif of NCS-1 and hippocalcin that resulted in both proteins remaining cytosolic, even at elevated Ca(2+) concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca(2+) concentration in Fura Red-loaded cells demonstrated the kinetics of the Ca(2+)/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of approximately 12 s after a short lag period when Ca(2+) was elevated. These results demonstrate that closely related Ca(2+) sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca(2+) signal transduction.

P/Q-type presynaptic calcium currents ( I pCa ) undergo activity-dependent facilitation during repetitive activation at the calyx of the Held synapse. We investigated whether neuronal calcium sensor 1 (NCS-1) may underlie this phenomenon. Direct loading of NCS-1 into the nerve terminal mimicked activity-dependent I pCa facilitation by accelerating the activation time of I pCa in a Ca 2+ -dependent manner. A presynaptically loaded carboxyl-terminal peptide of NCS-1 abolished I pCa facilitation. These results suggest that residual Ca 2+ activates endogenous NCS-1, thereby facilitating I pCa . Because both P/Q-type Ca 2+ channels and NCS-1 are widely expressed in mammalian nerve terminals, NCS-1 may contribute to the activity-dependent synaptic facilitation at many synapses.