Putative Calmodulin Binding Site Research Articles

Structural insights into trypanosomatid Mnk kinase orthologues (kMnks) suggest altered mechanism in the kinase domain

Mitogen-activated protein kinase (MAPK) interacting protein kinases (Mnk1 and Mnk2) mediated phosphorylation of the eukaryotic initiation factor eIF4E is an important translation initiation control, in Mnk-mediated oncogenic activity and other disease conditions. Thus, Mnk kinases are an important target for therapy. Trypanosomatids are a class of kinetoplastids, some of which are protozoan parasites and cause diseases in humans. While protein translation initiation is well understood in eukaryotes and prokaryotes, there is a lack of sufficient structural information of this process in trypanosomatids. Here, we report that trypanosomatids have one orthologue of Mnk kinase with low overall sequence homology but high homology in the kinase domain and an additional C-terminal domain containing putative calmodulin binding site(s). We show that while many of the domains and motifs are conserved, homology modeling/structure prediction, docking analysis and molecular dynamics simulation studies suggest that trypanosomatid kMnk kinases, kinase domains are present in DFG-in conformation as opposed to the auto-inhibited DFD-out conformation of un-phosphorylated human Mnk1. Furthermore, we observed that several regulatory features are different in trypanosomatid kMnk kinases. Our study indicates that mechanism and regulation in the kinase domain of trypanosomatid kMnks are likely to be altered, and that they can be important drug targets.

Interaction between IDH1 WT and calmodulin and its implications for glioblastoma cell growth and migration

Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas.

Insect odorant receptor trafficking requires calmodulin.

BackgroundLike most animals, insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators. Most insect olfactory neurons express an odorant-specific odorant receptor (OR) along with Orco, the olfactory co-receptor. Orco binds ORs and permits their trafficking to the dendrites of antennal olfactory sensory neurons (OSNs), where together, they are suggested to form heteromeric ligand-gated non-selective cation channels. While most amino acid residues in Orco are well conserved across insect orders, one especially well-conserved region in Orco’s second intracellular loop is a putative calmodulin (CaM) binding site (CBS). In this study, we explore the relationship between Orco and CaM in vivo in the olfactory neurons of Drosophila melanogaster.ResultsWe first found OSN-specific knock-down of CaM at the onset of OSN development disrupts the spontaneous firing of OSNs and reduces Orco trafficking to the ciliated dendrites of OSNs without affecting their morphology. We then generated a series of Orco CBS mutant proteins and found that none of them rescue the Orco-null Orco1 mutant phenotype, which is characterized by an OR protein trafficking defect that blocks spontaneous and odorant-evoked OSN activity. In contrast to an identically constructed wild-type form of Orco that does rescue the Orco1 phenotype, all the Orco CBS mutants remain stuck in the OSN soma, preventing even the smallest odorant-evoked response. Last, we found CaM’s modulation of OR trafficking is dependent on activity. Knock-down of CaM in all Orco-positive OSNs after OR expression is well established has little effect on olfactory responsiveness alone. When combined with an extended exposure to odorant, however, this late-onset CaM knock-down significantly reduces both olfactory sensitivity and the trafficking of Orco only to the ciliated dendrites of OSNs that respond to the exposed odorant.ConclusionsIn this study, we show CaM regulates OR trafficking and olfactory responses in vivo in Drosophila olfactory neurons via a well-conserved binding site on the olfactory co-receptor Orco. As CaM’s modulation of Orco seems to be dependent on activity, we propose a model in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odorant concentrations.

Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.

Gap junction regulation by calmodulin

Intracellular Ca(2+) activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca(2+)]i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely "invisible" in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca(2+)-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies.

The Stoichiometry of an SK Channel Peptide and Calmodulin

SK potassium channels modulate the cellular membrane potential in response to intracellular calcium. SK requires that calcium binds to the calcium sensor, calmodulin (CaM), to open. Comprised of four identical subunits, SK has at least four putative CaM binding sites; at least one site is encoded per carboxyl tail. Understanding how SK channels operate requires knowing how many CaMs bind to SK. Previous work has shown that in a crystal the SK peptide (SKp) forms a ‘2:2 complex' with CaM in saturating calcium. We have used a recombinant CaM binding SK peptide (SKp) without affinity tags to measure stoichiometry. Orthogonal approaches were used to measure the molecular mass of particles in solution and in the presence of 1 mM calcium. Light scattering results show that SKp and CaM form a complex with a molecular mass close to 29 kDa, which is consistent with a 1:1 SKp:CaM complex. Sedimentation equilibrium studies show an intermediate molecular weight consistent with alternate stoichiometries, but not likely present as a 2:2 complex. In sedimentation velocity measurements, the sedimentation rate of the complex that forms varies with the molar ratio of SKp:CaM. For example, when SKp and CaM are equimolar, van Holde-Weischet analyses yields a sedimentation constant of 2.50 s (s=1x10−13s). If CaM is in two fold molar excess over SKp, the sedimentation constant increases to 2.96 s. If SKp is in two fold molar excess over CaM, a distinct species forms with a sedimentation constant of 2.75 s. Finally, 2D spectrum analyses combined with Monte Carlo statistics support alternative stoichiometric ratios, but also exclude the formation of a ‘2:2 complex'. The data suggest these proteins have different modes of interaction in a dilute solution than occurs in a crystal.

Plexin, small GTPases and their interactions with the calcium binding protein, Calmodulin: a possible mechanism for calcium to influence cell motility

The plexin family is a group of transmembrane receptor proteins that are activated and function in cell motility by binding to small GTPases at their intracellular region. The interactions between plexin and the small GTPases such as Rac‐1 and Ras are well documented. The small GTPases regulate the activity of plexin by cooperating with extracellular semaphorin ligands to initiate a cellular response. However, there is evidence that the small GTPases are not the sole regulators of plexin. Plexin signaling is known to be sensitive to calcium and thus it is possible that a calcium binding protein, such as calmodulin, may have a role in regulating plexin's activity. We have found several putative calmodulin binding sites in plexins based on predictions from the calmodulin Target Database. Even if binding of calmodulin to plexin is not direct, but calmodulin is known to bind several of the plexin associated GTPases. Thus, it is possible that calmodulin regulates plexin by inhibiting the GTPases in their binding to plexin. In order to resolve the mode of calmodulin influence, we are currently studying the interaction of calmodulin with plexins and GTPases using isothermal titration calorimetry.

칼모듈린에 결합하는 대두 Ca2+-ATPase 2 (SCA2)의 분리 및 특성 분석

We previously reported the isolation and characterization of a gene, SCA1 (for soybean Ca²?-ATPase 1), encoding a calmodulin-regulated Ca²?-ATPase that is located in the plasma membrane in soybean. Here, a Ca²?-ATPase designated as SCA2 was isolated from soybean. The two Ca²?-ATPases, SCA1 and SCA2, share a remarkably high degree of similarity (78%). Ten transmemebrane domains were predicted by hydropathy analysis. Using gel overlay assays, CaM was found to bind to SCA2 in a Ca²?-dependent manner. Southern blot analysis revealed the presence of two copies of the Ca²?-ATPase gene in the soybean genome. An N-terminal truncation mutant that deletes sequence through the putative calmodulin binding site was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca²? pumps. Our results indicate that SCA2 is structurally highly conserved with type IIB Ca²? pumps in plants.

Tat peptide‐calmodulin binding studies and bioinformatics of HIV‐1 protein–calmodulin interactions

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.

Abstract 4226: Curcumin induced CaSR stimulates calpain autolysis in colon cancer

Abstract Colon cancer is the fourth most common form of cancer in the United States and the third leading cause of cancer-related death in the Western world. Progression to colon cancer is distinguished with a loss of the colonic calcium sensing receptor (CaSR). It has been observed that calcium-dependent proteolytic enzyme, Calpain (Capn) overexpressed in colon cancer may contribute to its metastasis. We attempted to identify role of calcium sensing receptor CaSR on regulation of capn activity and its influence on calmodulin (CaM) homeostasis. We have also investigated the role of curcumin in activation of CaSR. Curcumin (1μM and 20μM) increases expression (qPCR) and protein (2-3 fold) of the CaSR in wild type intestinal cells (RKO; wild type APC, wild type β-catenin) and cells with truncated APC (HT-29, SW480). Transient transfection of siRNA (200 nM) duplex against CaSR reduced CaSR protein expression and prevented reduction of Capn activity after 3 mM Ca2+ challenge (340+/-24 Units, p<0.05 n=4). Western blotting of HT-29 cell lysates after 3 mM Ca2+ challenge demonstrated no change in amount of Calpain-4 (Capn4, small subunit of Capn essential for its activity) but a 6 fold increase in CaM. Bioinformatic analysis of Capn4 revealed a presence of putative CaM binding site. Using circular dichroism, isothermal titration calorimetry and nuclear magnetic resonance it was confirmed that Capn4 binds to CaM. This is a first report of its kind demonstrating interaction of two EF-hand proteins. Capn after 30 min incubation with CaM demonstrated a 3 fold increase in autolysed Capn and 5 fold reduction in 75 kDa large-subunit of Capn. CaM antagonists W7 or W13 (100 μM) prevented CaSR-mediated decrease in Calpain activity (p<0.05, n=4). We hereby conclude that curcumin will increase CaSR expression and that calcium will stimulate the CaSR to increase calmodulin in colonic adenocarcinoma to produce Capn autolysis which will reduce Capn activity. We speculate that CaSR-mediated reduction in Capn activity may be an important determinant of calcium chemoprevention of colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4226. doi:10.1158/1538-7445.AM2011-4226

Disease‐causing mutation R1185C alters phosphorylation of WNK4 by SGK1 via a calmodulin binding site

WNK4 is a protein serine/threonine kinase, which is linked to familial hyperkalemic hypertension (FHH). In this study, we evaluated 2 putative calmodulin (CaM) binding sites in WNK4 (amino‐acids 502–525 and 1175–1194) as predicted by a network based CaM target database. The first CaM binding site is close to a cluster of FHH‐causing mutations (E562K, D564A and Q565E) whereas the second contains a FHH‐causing mutation (R1185C) and S1190, a SGK1 phosphorylation site. The two GST‐fusion proteins containing CaM binding sites of WNK4, GST‐WNK4a (492–552) and GST‐WNK4b (1163–1212), exhibited CaM binding ability using pull down assay with CaM‐agarose. Reducing a positive charge in R1185C mutation and introducing a negative charge in phospho‐mimicking S1190D mutation resulted in a reduction of CaM binding ability in GST‐WNK4b. On the other hand, in vitro phosphorylation of GST‐WNK4b by SGK1 was inhibited by CaM in a Ca2+ dependent manner and it was less Ca2+dependent for the R1185C mutant. These results indicate Ca2+‐CaM is involved in the regulation of WNK4 and this regulation may be altered under FHH condition.

KCNE4 Juxtamembrane Region Interacts with Calmodulin and is Necessary for KCNQ1 Modulation

Voltage-gated potassium (KV) channels are modulated by the KCNE family of single transmembrane proteins. A membrane-based yeast two-hybrid screen to discover KCNE4 interacting proteins identified calmodulin (CaM) as a candidate. Previous studies demonstrated that CaM binding to KCNQ1 is required for functional expression of KCNQ1-KCNE1 channels in vitro, and increasing concentrations of intracellular calcium stimulate KCNQ1-KCNE1 channels in Xenopus oocytes in the presence of wild-type CaM but not mutant CaM that cannot bind calcium. We have tested the functional consequences of the interaction between KCNE4 and CaM with the hypothesis that KCNE4 modulation of KV currents may depend on its interaction with CaM. We validated the biochemical interaction between KCNE4 and CaM using CaM-agarose pull-down, and tested KCNE4 mutants that targeted putative CaM binding sites. Mutation of a juxtamembrane site (L[69-72]A) exhibited near complete disruption of CaM binding, whereas biotinylation studies performed in CHO cells confirmed expression of the mutant protein at the cell surface. The ability of L[69-72]A to modulate KCNQ1 was then studied using whole-cell patch clamp recording to determine if functional consequences accompany the loss of CaM binding. Wild-type KCNE4 completely inhibits potassium current in CHO cells transiently co-transfected with KCNQ1, but cells co-expressing KCNQ1 with L[69-72]A exhibited KCNQ1-like currents. Mean (± SEM) current density (measured during step to +60 mV from holding potential of −80 mV) in cells expressing KCNQ1 alone was 37.0 ± 4.25, not significantly different from cells co-expressing KCNQ1 with L[69-72]A (32.1 ± 3.3), but significantly different from cells co-expressing KCNQ1 with wild-type KCNE4 (3.3 ± 0.35). These studies suggest that a juxtamembrane region in KCNE4 is critical for its interaction with CaM and is necessary for modulation of KCNQ1.

Aquaporin 6 binds calmodulin in a calcium-dependent manner

Aquaporin 6 (AQP6) is an anion channel that is expressed primarily in acid secreting α-intercalated cells of the kidney collecting duct. In addition, AQP6 anion channel permeability is gated by low pH. Inspection of the N-terminus of AQP6 revealed a putative calmodulin binding site. AQP6-expressing CHO-K1 cell lysates were mixed with calmodulin beads and AQP6 was pulled down in the presence of calcium. Mutagenesis of the N-terminal calmodulin binding site in full length mouse AQP6 resulted in a loss of calmodulin binding activity. Mouse and human AQP6 calmodulin binding site peptides bound dansyl-calmodulin with a dissociation constant of approximately 1μM. The binding of AQP6 to calmodulin may be an important key to determining the physiological role of AQP6 in the kidney.

The potatoStLTPa7gene displays a complex Ca2+‐associated pattern of expression during the early stage of potato–Ralstonia solanacearuminteraction

Although nonspecific lipid transfer proteins (nsLTPs) are widely expressed during plant defence responses to pathogens, their functions and regulation are not fully understood. In this article, we report the isolation of a cDNA for the new nsLTP, StLTPa7, from cultivated potato (Solanum tuberosum) infected with Ralstonia solanacearum. The cDNA was predicted to encode a type 1 nsLTP containing an N-terminal signal sequence and possessing the characteristic features of nsLTPs. A phylogenetic analysis showed that the encoded amino acid sequence of the nsLTP was similar to those of other previously reported plant nsLTPs, which contain a putative calmodulin-binding site consisting of approximately 12 highly conserved amino acid residues. The expression of the StLTPa7 gene was studied during the early stages of potato-R. solanacearum interaction using real-time quantitative polymerase chain reaction (qRT-PCR) and Northern analyses, and a complex calcium (Ca2+)-associated pattern of expression was observed with the following features: (i) transcripts of the StLTPa7 gene were systemically up-regulated by infection with R. solanacearum; (ii) the StLTPa7 gene was stimulated by salicylic acid, methyl jasmonate, abscisic acid and Ca2+; (iii) qRT-PCR showed that, during the early stage of R. solanacearum infection, nsLTP transcripts accumulated over a time course that paralleled that of Ca2+ accumulation, detected using environmental scanning electron microscopy and energy-dispersive X-ray (EDAX) spectrometry; and (iv) the Ca2+ channel blocker, ruthenium red, partially blocked R. solanacearum-induced StLTPa7 expression. This report represents the first use of EDAX analysis to establish a synchrony between Ca2+ accumulation and nsLTP expression in response to potato-R. solanacearum interactions. Collectively, these results suggest that StLTPa7 may be a pathogen- and Ca(2+)-responsive plant defence gene.

Calmodulin, a regulatory partner of the estrogen receptor alpha in breast cancer cells

Although calmodulin (CaM) interaction with estrogen receptor alpha (ERalpha) has been known for more than two decades, it is only recently that the molecular mechanism of CaM-mediated regulation of ERalpha is beginning to emerge. Others and we have identified a putative calmodulin binding site (P(295)LMIKRSKKNSLALSTADQMVS(317)) in ERalpha, at the boundary between the hinge and the ligand binding domain. ERalpha mutations affecting its association with CaM have been reported to generate high basal, estrogen-independent transactivation activity, indicating that the P(295)-T(317) sequence has an inhibitory function. Moreover, we found that a synthetic peptide (ERalpha17p: P(295)-T(311)) containing residues crucial for CaM binding exerts estrogenic effects on breast carcinoma cells. Finally, computer-aided conformational studies revealed that the CaM binding site might associate with a region located downstream in ERalpha (the beta turn/H4 region), this association likely resulting in an auto-inhibitory folding of the receptor. Thus, we propose as a hypothesis that CaM acts as a positive regulator by relieving this ERalpha auto-inhibition.

Aquaporin 0−Calmodulin Interaction and the Effect of Aquaporin 0 Phosphorylation

Aquaporin 0 (AQP0), also known as major intrinsic protein of lens, is the most abundant membrane protein in the lens and it undergoes a host of C-terminally directed posttranslational modifications. The C-terminal region containing the major phosphorylation sites is a putative calmodulin-binding site, and calmodulin has been shown to regulate AQP0 water permeability. The purpose of the present study was to elucidate the role of AQP0 phosphorylation on calmodulin binding. AQP0 C-terminal peptides were synthesized with and without serine phosphorylation on S231 and S235, and the ability of these peptides to bind dansyl-labeled calmodulin and the calcium dependence of the interaction was assessed using a fluorescence binding assay. The AQP0 C-terminal phosphorylated peptides were found to have 20-50-fold lower affinities for calmodulin than the unphosphorylated peptide. Chemical cross-linking studies revealed specific sites of AQP0-calmodulin interaction that are significantly reduced by AQP0 phosphorylation. These data suggest that AQP0 C-terminal phosphorylation affects calmodulin binding in vivo and has a role in regulation of AQP0 function.

Binding property of avian skeletal muscle ryanodine receptor isoforms with dihydropyridine receptor and calmodulin

Ca(2+) release during excitation-contraction coupling in avian skeletal muscle is controlled by two ryanodine receptor isoforms, alphaRYR and betaRYR. Two other proteins, dihydropyridine receptor (DHPR) and calmodulin (CaM), have been shown to play important roles in regulating the RYR channel activity. In the current study, we measured the protein contents of DHPR and RYR in turkey skeletal muscle and obtained a ratio of 1:1 between DHPR and alphaRYR which suggests that only a subpopulation of alphaRYR is interacting with DHPR. Two CaM derivatives, the photoactivable crosslinking probe [(125)I]-Bz-CaM and metabolically labeled probe [(35)S]CaM, were used to study the interaction between CaM and RYR isoforms in turkey skeletal muscle. The alphaRYR and betaRYR displayed a marked difference in their CaM binding behavior. At a Ca(2+) concentration of 200 microM, CaM bound to both isoforms at a ratio of one CaM molecule per one RYR subunit. At a Ca(2+) concentration of <10 nM, CaM bound primarily to alphaRYR and the binding affinity was significantly lower than that at micromolar level of Ca(2+) concentration. Cloning and sequencing of putative CaM binding sites in alphaRYR and betaRYR suggests that differences in primary structures of the CaM binding sites of each RYR isoform may contribute to the differential CaM binding behavior of alphaRYR and betaRYR.

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

Molecular Basis of Calmodulin Binding to Cardiac Muscle Ca2+ Release Channel (Ryanodine Receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.