Calmodulin-binding Domain Research Articles

Identification of three distinct calmodulin‐binding domains in the G Protein‐coupled Estrogen Receptor

The novel G protein‐coupled estrogen receptor (GPER/GPR30) is being increasingly identified as an important GPCR in many organ system. Calmodulin (CaM) is a ubiquitous Ca2+ transducer that is required for the activities of numerous cellular proteins. Here we show that CaM coimmunoprecipitates with GPER in primary vascular endothelial cells upon stimulation with 17β‐estradiol or Ca2+‐elevating ionophore. To identify the CaM‐binding sites in GPER/GPR30, we have developed a series of novel FRET‐based biosensors whose responses allow identification and highly quantitative characterization of CaM‐binding properties of CaM‐binding domains in GPER/GPR30. Three separate CaM‐binding domains with distinct dissociation constants and Ca2+ sensitivities for complex formation with CaM were found. The functional implications of CaM‐GPER interactions are explored.

Impaired intracellular Ca2+ signaling alters the activity of growth promoting signaling molecules in Ryr2ADA/ADA mice

Substitution of three amino acid residues in the calmodulin (CaM) binding domain of the cardiac ryanodine receptor ion channel (W3587A, L3591D, F3603A, RyR2ADA) impairs CaM inhibition of RyR2, alters intracellular Ca2+ handling and leads to rapid increase in heart size and death of Ryr2ADA/ADA mice within about 2 weeks of birth (Yamaguchi et al., J Clin Invest 117: 1344–1353, 2007). To elucidate mechanisms by which cardiac heart size is increased, we determined the protein and phosphorylation levels of signaling molecules in hearts of 1‐day and 10‐day old Ryr2ADA/ADA and wild type mice. Immunoblots showed a significant increase in phosphorylation levels of mTOR at Ser2448, p70 S6 kinase at Thr389, S6 ribosomal protein at Ser235/236 and 240/244 and eukaryotic translation initiation factor 4B (eIF4B) at Ser422 with no major changes in protein expression levels. Phosphorylation of AMP‐activated protein kinase (AMPK) at Thr172 was decreased, whereas phosphorylation of insulin receptor substrate 1 (IRS1) at Ser612, p38 mitogen‐activated protein kinase (p38MAPK) at Thr180/Tyr182, phosphoinositide kinase 1 (PDK1) at Ser241 and p90 ribosomal S6 kinases (p90RSKs) at Ser380 and Thr 359/Ser 363 were all increased at postnatal day 10 in Ryr2ADA/ADA mice. The results indicate that impaired CaM regulation of RyR2 alters the activity of multiple signaling pathways regulating cardiac metabolism and growth. Supported by NIH.

Ca2+ /S100 proteins regulate HCV virus NS5A-FKBP8/FKBP38 interaction and HCV virus RNA replication

FKBP8/FKBP38 is a unique FK506-binding protein with a C-terminal membrane anchor and localizes at the outer membranes of mitochondria and the endoplasmic reticulum. Similar to some immunophilins, such as FKBP51, FKBP52 and Cyclophilin 40, FKBP8/FKBP38 contain a putative Calmodulin-binding domain and a tetratricopeptide-repeat (TPR) domain for the binding of Hsp90. Both Hsp90 and the non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) interact specifically with FKBP8/FKBP38 through its TPR domain, and the ternary complex formation plays a critical role in HCV RNA replication. The goal of this study is to evaluate that the host factor inhibits the ternary complex formation and the replication of HCV in vitro and in vivo. S100 proteins, FKBP38, FKBP8, HCV NS5A, Hsp90, and calmodulin were expressed in E.coli and purified. In vitro binding studies were performed by GST pull-down, S-tag pull-down and surface plasmon resonance analyses. The effect of S100 proteins on HCV replication was analysed by Western blotting using an HCV NS3 antibody following transfection of S100 proteins into the HCV replicon harbouring cell line (sO cells). In vitro binding studies showed that S100A1, S100A2, S100A6, S100B and S100P directly interacted with FKBP8/FKBP38 in a Ca(2+) -dependent manner and inhibited the FKBP8/FKBP38-Hsp90 and FKBP8/FKBP38-NS5A interactions. Furthermore, overexpression of S100A1, S100A2 and S100A6 in sO cells resulted in the efficient inhibition of HCV replication. The association of the S100 proteins with FKBP8/FKBP38 provides a novel Ca(2+) -dependent regulatory role in HCV replication through the NS5A-host protein interaction.

The plasma membrane calcium pump in health and disease

The Ca(2+) ATPases of the plasma membrane (PMCA pumps) export Ca(2+) from all eukaryotic cells. In mammals they are the products of four separate genes. PMCA types 1 and 4 are distributed ubiquitously; PMCA types 2 and 3 are restricted to some tissues, the most important being the nervous system. Alternative splicing at two sites greatly increases the number of pump isoforms. The two ubiquitous isoforms are no longer considered as only housekeeping pumps as they also perform tissue-specific functions. The PMCAs are classical P-type pumps, their reaction cycle repeating that of all other pumps of the family. Their 3D structure has not been solved, but molecular modeling on SERCA pump templates shows the essential structural pattern of the latter. PMCAs are regulated by calmodulin, which interacts with high affinity with their cytosolic C-terminal tail. A second calmodulin-binding domain with lower affinity is present in some splicing variants of the pump. The PMCAs are essential to the regulation of cellular Ca(2+), but the all-important Ca(2+) signal is ambivalent: defects in its control generate various pathologies, the most thoroughly studied being those of genetic origin. Genetic defects of PMCA function produce disease phenotypes: the best characterized is a form of deafness in mice and in humans linked to PMCA2 mutations. A cerebellar X-linked human ataxia has recently been found to be caused by a mutation in the calmodulin-binding domain of PMCA3.

Unstructured to structured transition of an intrinsically disordered protein peptide in coupling Ca 2+ -sensing and SK channel activation

Most proteins, such as ion channels, form well-organized 3D structures to carry out their specific functions. A typical voltage-gated potassium channel subunit has six transmembrane segments (S1-S6) to form the voltage-sensing domain and the pore domain. Conformational changes of these domains result in opening of the channel pore. Intrinsically disordered (ID) proteins/peptides are considered equally important for the protein functions. However, it is difficult to explore the structural features underlying the functions of ID proteins/peptides by conventional methods, such as X-ray crystallography, because of the flexibility of their secondary structures. Unlike voltage-gated potassium channels, families of small- and intermediate-conductance Ca(2+)-activated potassium (SK/IK) channels with important roles in regulating membrane excitability are activated exclusively by Ca(2+)-bound calmodulin (CaM). Upon binding of Ca(2+) to CaM, a 2 × 2 structure forms between CaM and the CaM-binding domain. A channel fragment that connects S6 and the CaM-binding domain is not visible in the protein crystal structure, suggesting that this fragment is an ID fragment. Here we show that the conformation of the ID fragment in SK channels becomes readily identifiable in the presence of NS309, the most potent compound that potentiates the channel activities. This well-defined conformation of the ID fragment, stabilized by NS309, increases the channel open probability at a given Ca(2+) concentration. Our results demonstrate that the ID fragment, itself a target for drugs modulating SK channel activities, plays a unique role in coupling Ca(2+) sensing by CaM and mechanical opening of SK channels.

Nuclear magnetic resonance imaging of tumour growth and neovasculature performance in vivo reveals Grb7 as a novel antiangiogenic target

Development of neovasculature is a necessary requirement for tumour growth and it provides additional opportunities for therapeutic intervention. However, current antiangiogenic therapies have limited efficacy, mostly because of the development of resistance. Hence, characterization of new antiangiogenic molecular targets is of considerable clinical interest. We report that a calmodulin-binding domain (CaM-BD) deletion mutant of the growth factor receptor bound protein 7 (Grb7) (denoted Grb7Δ) impairs tumour growth and associated angiogenesis in vivo. We implanted glioma C6 cells in rat brains transfected with an enhanced yellow fluorescent protein (EYFP) chimera of Grb7∆, its EYFP-Grb7 wild type counterpart, and EYFP alone. We systematically followed intracerebral growth of the tumours, their cellularity and the functional performance of tumour-associated microvasculature using magnetic resonance imaging, including anatomical T1W and T2W images and functional diffusion and perfusion parameters. Tumours grown from implanted C6 cells expressing EYFP-Grb7Δ developed slower, became smaller and presented lower apparent cellularity than those derived from cells expressing EYFP-Grb7 and EYFP. Vascular perfusion measurements within tumours derived from EYFP-Grb7∆-expressing cells showed significantly lower cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) values. These findings were independently validated by histological and immunohistochemical techniques. Taken together, these findings confirm that the CaM-BD of Grb7 plays an important role in tumour growth and associated angiogenesis in vivo, thus identifying this region of the protein as a novel target for antiangiogenic treatment.

A general framework to characterize inhibitors of calmodulin: Use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains

The prominent role of Ca2+ in cell physiology is mediated by a whole set of proteins involved in Ca2+-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells.In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM.Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent.The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Resonance assignments and secondary structure of calmodulin in complex with its target sequence in rat olfactory cyclic nucleotide-gated ion channel

Calmodulin (CaM), the primary receptor for intracellular Ca(2+), regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Olfactory cyclic nucleotide-gated ion channels (OLF channels) mediate olfactory transduction in olfactory receptor neurons. The opening of OLF leads to a rise in cytosolic concentration of Ca(2+), upon binding to Ca(2+), CaM disrupts the open conformation by binding to the CaM-binding domain in the N-terminal region and triggers the close mechanism. In order to unravel the regulatory role of CaM from structural point of view, NMR techniques were used to characterize the structure of CaM in association with the CaM binding domain of rat OLF channel (OLFp, 28 residues). Our data indicated that two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not inter-exchangeable within the NMR time scale. Here, we report the full backbone and side chain resonance assignments of these two complexes of CaM/OLFp.

Regulation of Calcineurin by Domain-Specific Interactions with Calmodulin

Calcineurin (CaN) is a heterodimeric Ser/Thr phosphatase. Calcineurin regulates diverse biological functions, from T-cell activation to critical events in early heart development. The enzymatic activity of the A subunit (CaNA) is regulated by direct binding of Ca2+ to its B subunit (CaNB), and Ca2+ binding of the two-domain calcium-binding protein calmodulin (CaM) that is required for full activity. CaNA contains an auto-inhibitory domain that prevents full activation of CaN even when CaNB is Ca2+ saturated. Interactions between CaM and CaN occur through a CaM-binding domain (CaMBD). This sequence (391-414 of α CaNA) is a BAA-motif (basic amphipathic alpha-helix) in a region that becomes disordered when CaNB binds calcium. (Ca2+)4-CaM has picomolar affinity for CaN, as determined by thermodynamic linkage (O'Donnell et al, Proteins 2011). The Creamer Lab (Rumi-Masante et al, JMB 2012) has shown that CaM-binding induces secondary structuring of the full regulatory region of CaNA that extends beyond this CaMBD. We have also determined that calcium binding to CaM bound to the CaMBD is sequential, with the C-domain sites saturating at lower calcium than the N-domain sites. Thus, we are investigating a hypothesis that the mechanism of calcium-induced activation of CaN requires at least three steps that utilize the two domains of CaM (N and C) differently. We are using “knockout” mutants of CaM with modifications in one or more calcium-binding sites, and monitoring their binding to biosensor proteins containing the CaMBD embedded between auto-fluorescent proteins. Without CaM binding the fluorescent proteins undergo FRET, while CaM binding to the BAA motif reduces FRET efficiency and allows calculations to be made on CaM-biosensor interaction kinetics. We will report domain-specific effects of these mutations on recognition of this region. Support: NIH R01 GM57001, UI Helen Johnson Scholar Award, American Heart Association 12GRNT12050395.

Cardiac Hypertrophy Associated with Impaired Regulation of Type2 Ryanodine Receptor Calcium Channel by Calmodulin and S100A1

The cardiac ryanodine receptor ion channel (RyR2) is inhibited in vitro by calmodulin (CaM). Simultaneous substitution of three amino acid residues in the CaM binding domain (W3587A/L3591D/F3603A, RyR2ADA) leads to loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca2+ concentrations in vitro and cardiac hypertrophy and heart failure in mice. To address whether elimination of CaM inhibition at diastolic or systolic Ca2+ causes cardiac hypertrophy, a second mutant mouse was prepared with a single amino acid substitution (L3591D, RyR2D) in the CaM binding domain. In single channel measurements, RyR2D lost CaM inhibition at diastolic but not systolic Ca2+, and lost inhibition by the small Ca2+ binding protein S100A1 at both diastolic and systolic Ca2+. In contrast to Ryr2ADA/ADA mice, Ryr2D/D mice had a normal lifespan and cardiac contractility. In 6-month old Ryr2D/D mice, heart-to-body weight ratio increased by 8% with a two-fold upregulation of atrial natriuretic peptide mRNA levels and a 40% decrease in RyR2 content. Differences between mutant and wild-type mice were more prominent in mice subjected to 4 weeks pressure overload using transverse aortic constriction. The results contrast those of Ryr2ADA/ADA mice that have an impaired cardiac contractile performance and die at ∼2 weeks after birth, and suggest that CaM inhibition of RyR2 at systolic Ca2+ is important for maintaining normal cardiac function. Supported by NIH, AHA and NSF.

Differential Impact of Distinct Long Qt Syndrome-1 (LQTS-1) C-Terminus Mutations on KCNQ1-KCNE1 Channel Trafficking and Gating

KCNQ1/KCNE1 subunits together generate the slowly activating, delayed rectifier potassium current, IKs, important for human cardiac repolarization. Mutations throughout KCNQ1 lead to long QT syndrome-1 (LQTS-1), which predisposes patients to lethal ventricular arrhythmias. Many LQTS-1 mutations map to KCNQ1 C-terminus, but in many cases the mechanistic bases for their pathophysiological effects in heart are unknown. We compared the functional impact and mechanistic bases of six LQTS-1 mutations in distinct regions of KCNQ1 C-terminus that have been implicated in: PIP2 or calmodulin binding domains (R555H, L619M). Optical assays for channel surface density and electrophysiological recordings were carried out in Chinese Hamster Ovary (CHO)cells. All KCNQ1 mutants, except L619M, displayed a significant decrease in channel surface density, which was either partially (R366W, V524G, R539W,G589D) or fully (R555H) rescued by wild-type KCNQ1. G589D and L619M alone yielded no currents, while all other mutations moderately reduced current amplitude. When co-expressed with wild-type KCNQ1, G589D current amplitude was fully rescued whereas L619M exerted a dominant negative effect. When co-expressed with KCNE1: all mutants displayed reduced current amplitude compared to control, albeit to different extents; all except L619M displayed the slowly activating signature of IKs; three mutants displayed a large rightward shift in the activation curve which was either partially (R366W, R555H) or fully (R539W) recovered with wild-type KCNQ1. The data reveal a surprising heterogeneity of trafficking and gating mechanisms underlie KCNQ1 C-terminus mutations. Such information is essential for developing potential targeted therapies for LQTS-1 patients. We further introduce the first study of L619M, an unusual KCNQ1 mutant that trafficks normally to the cell surface, but is essentially non-functional and exerts a strong dominant-negative effect on wild-type KCNQ1 channels.

Wild-Type Sodium Channels and ‘Atypical' Brugada Syndrome Mutants Interact through C-Terminal Region

Introduction: Brugada syndrome (BrS) is an inherited cardiac disorder that can be caused by mutations in the cardiac sodium channel gene resulting in a reduction of sodium currents. However, several ‘atypical’ sodium channel mutations identified in patients show miminal biophysical defects questioning how such mutants may produce a BrS phenotype. We have previously demonstrated that when “atypical” BrS mutants were co-expressed with wild-type channels (WT) mimicking the heterozygous patient genotype, this lead to a dramatic reduction in sodium currents thereby explaining the disease phenotype. Here we hypothesized that a direct interaction between atypical BrS mutants and WT is mediated via the calmodulin-binding IQ domain of the channel protein. Methods: ‘Atypical’ BrS mutations with minimal biophysical defects were co-expressed with either WT or WT channels containing a mutated calmodulin binding IQ motif (IQ/AA) in HEK293 cells. BrS mutations from all intracellular loops were studied. Biophysical properties of mutants were investigated by patch clamp. Co-immunoprecipitation and cell surface biotinylation were performed to assess interaction and channel location. Results: BrS mutants co-expressed with IQ/AA had current amplitudes restored to control levels. This abrogated any loss-of-current phenotype we observed when the “atypical” mutants were co-expressed with WT. Cell surface biotinylation showed a significant reduction of both WT and mutant channels at the plasma membrane on co-expression, while surface expression was restored for both channels when mutants were co-expressed with the IQ/AA construct. Importantly, while co-immunoprecipitation experiments demonstrated interactions between WT and “atypical” mutant channels, IQ/AA failed to interact with atypical BrS mutants. Conclusions: Our data suggest that ‘atypical’ BrS mutations suppress both mutant and WT channels expression at the cell surface via interactions mediated by the calmodulin-binding (IQ) domain of the cardiac sodium channel.

Genome-wide identification, characterization, and expression analysis of the MLO gene family in Cucumis sativus

Mildew resistance locus o (MLO) is a plant-specific seven-transmembrane (TM) gene family. Several studies have revealed that certain members of the MLO gene family mediate powdery mildew susceptibility in three plant species, namely, Arabidopsis, barley, and tomato. The sequenced cucumber genome provides an opportunity to conduct a comprehensive overview of the MLO gene family. Fourteen genes (designated CsMLO01 through CsMLO14) have been identified within the Cucumis sativus genome by using an in silico cloning method with the MLO amino acid sequences of Arabidopsis thaliana and rice as probes. Sequence alignment revealed that numerous features of the gene family, such as TMs, a calmodulin-binding domain, peptide domains I and II, and 30 important amino acid residues for MLO function, are well conserved. Phylogenetic analysis of the MLO genes from cucumber and other plant species reveals seven different clades (I through VII). Three of these clades comprised MLO genes from A. thaliana, rice, maize, and cucumber, suggesting that these genes may have evolved after the divergence of monocots and dicots. In silico mapping showed that these CsMLOs were located on chromosomes 1, 2, 3, 4, 5, and 6 without any obvious clustering, except CsMLO01. To our knowledge, this paper is the first comprehensive report on MLO genes in C. sativus. These findings will facilitate the functional characterization of the MLOs related to powdery mildew susceptibility and assist in the development of disease resistance in cucumber.

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain

Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595-CFP and K4269-YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.

Structural Characterization of the Interaction of Human Lactoferrin with Calmodulin

Lactoferrin (Lf) is an 80 kDa, iron (Fe3+)-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf’s interaction with calmodulin (CaM), a ubiquitous, 17 kDa regulatory calcium (Ca2+)-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 kDa), TROSY-based NMR techniques were employed to structurally characterize Ca2+-CaM when bound to intact apo-Lf. Both CaM’s backbone amides and the ε-methyl group of key methionine residues were used as probes in chemical shift perturbation and cross-saturation experiments to define the binding interface of apo-Lf on Ca2+-CaM. Unlike the collapsed conformation through which Ca2+-CaM binds the CaM-binding domains of its classical targets, Ca2+-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca2+-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM’s primary binding domain. Secondary interfaces play a key role in the target’s response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes.

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+•Calmodulin Displays a Unique Binding Mode

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca(2+)-depletion sensor STIM1. This is followed by a fast Ca(2+)·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp(76) of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.

An Elevation in Physical Coupling of Type 1 Inositol 1,4,5-Trisphosphate (IP 3 ) Receptors to Transient Receptor Potential 3 (TRPC3) Channels Constricts Mesenteric Arteries in Genetic Hypertension

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C–coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP 3 ) that activates sarcoplasmic reticulum IP 3 receptors. In cerebral artery myocytes, IP 3 receptors release sarcoplasmic reticulum Ca 2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP 3 receptors control vascular contractility in systemic arteries and IP 3 R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP 3 R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP 3 -induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP 3 receptor binding domain peptide, an IP 3 R-TRP physical coupling inhibitor, reduced IP 3 -induced cation current and ET-1–induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca 2+ -ATPase blocker, did not alter ET-1–stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP 3 R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP 3 R1 to TRPC3 channel molecular coupling augments ET-1–induced vasoconstriction during hypertension.

Cyclin-dependent kinase 5 is a calmodulin-binding protein that associates with puromycin-sensitive aminopeptidase in the nucleus of Dictyostelium

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 (132LLINRKGELKLADFGLARAFGIP154) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP.

Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8

Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca2+ signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca2+ signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca2+ ions. These two lobes have differing affinities for Ca2+, and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca2+]i. We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca2+-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca2+ binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca2+-CaM activation of AC1 and AC8, which may underpin their different physiological roles.

Resonance Assignments of Calmodulin Complexed with the Calmodulin-Binding Domain of Olfactory Nucleotide Gated Ion Channel

Resonance Assignments of Calmodulin Complexed with the Calmodulin-Binding Domain of Olfactory Nucleotide Gated Ion Channel