Calneuron I inhibits Ca channel activity in bovine chromaffin cells

Calneuron I (CalnI) is a calmodulin-like protein that contains two functional EF-hand motifs at the N-terminal and a hydrophobic segment at the C-terminal. CalnI was cloned from the adult rat cortex and fused with GFP at its N-terminal. When expressed in bovine chromaffin cells, wild-type CalnI was localized at the plasma membrane. However, a mutant that lacked the hydrophobic segment was localized in the cytosol and nucleus, while a Ca²⁺-binding-deficient mutant was found in the cytosol and at the plasma membrane. Evaluation using the whole-cell patch-clamp technique revealed that Ca²⁺ currents were inhibited by both wild-type CalnI and the Ca²⁺-binding-deficient mutant. When the bovine N-type Ca²⁺ channel was expressed in 293T cells, Ca²⁺ currents were mostly inhibited by co-expression of CalnI, but not by the mutant without the hydrophobic tail. These results suggest that CalnI attenuates Ca²⁺ channel activity and that its subcellular localization is important for this effect.     

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.     

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.     

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.     

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca²⁺]_i) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca²⁺]_i from 118 ± 21 to 440 ± 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL’s epileptogenic process. Q. Zhang and X. Lai contributed equally to this article.     

1H, 15N, and 13C chemical shift assignments of calcium-binding protein 1 (CaBP1)

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP₃Rs) and a variety of voltage-gated Ca²⁺ channels in the brain. We report complete NMR chemical shift assignments of Ca²⁺-free CaBP1 (residues 1–167, BMRB no. 15197).     

1H, 15N, and 13C chemical shift assignments of calcium-bound calcium-binding protein 1 (CaBP1)

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP₃Rs) and a variety of voltage-gated Ca²⁺ channels in the brain. We report complete NMR chemical shift assignments of Ca²⁺-bound CaBP1 (residues 1–167, BMRB no. 15623).     

Setagin and secretagogin-R22: Posttranscriptional modification products of the secretagogin gene

We describe two new variants of the recently identified hexa-EF-hand calcium binding protein secretagogin. The first variant (secretagogin-R22) is characterized by one single amino acid exchange (Q/R) at codon 22, most likely due to RNA editing. The second variant of secretagogin (setagin) consists of 49 amino acids. Due to a frame shift, only the first 27 amino acids are identical to secretagogin. We demonstrate that this protein truncation results in complete loss of the calcium binding capacity. Setagin expression was found in considerable amounts in the pancreas whereas secretagogin and secretagogin-R22 were also found in the central nervous system and organs containing neurendocrine cells.     

Statistical analysis of structural characteristics of protein Ca<Superscript>2+</Superscript>-binding sites

To better understand the biological significance of Ca²⁺, we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca²⁺-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 ± 2 vs. 7 ± 1), fewer protein ligands (4 ± 2 vs. 6 ± 1), and more water ligands (2 ± 2 vs. 1 ± 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H₂O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H₂O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 ± 1 vs. 3 ± 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix–loop–helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca–O–C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca²⁺-binding proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Michael Kirberger, Xue Wang, and Hai Deng contributed equally to this article.     

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity

Up to now, all reported Ca²⁺-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca²⁺ sensitivity was lower than aequorin. Results of pK_a calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.     

Structural basis for diversity of the EF-hand calcium-binding proteins

The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca2+-binding and target interaction properties. Here, our current understanding of the Ca2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11-14 amino acid residues in the Ca2+-binding loop are analyzed within the framework of the recently proposed two-step Ca2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca2+-binding loops in the two-EF-hand domain. This structure, named EFbeta-scaffold, defines the position of the bound Ca2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca2+-binding loop. It is proposed that the nature of the first ligand of the Ca2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca2+-induced conformational change in the EF-hand proteins.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

The DxDxDG motif for calcium binding: multiple structural contexts and implications for evolution

Calcium ions regulate many cellular processes and have important structural roles in living organisms. Despite the great variety of calcium-binding proteins (CaBPs), many of them contain the same Ca(2+)-binding helix-loop-helix structure, referred to as the EF-hand. In the canonical EF-hand, the loop contains three calcium-binding aspartic acid residues, which form the DxDxDG sequence motif, and is flanked by two alpha-helices. Recently, other CaBPs containing the same motif, but lacking one or both helices, have been described. Here, structural motif searches were used to analyse the full diversity of structural context in the known set of DxDxDG-containing CaBPs, including those where the structural resemblance of a given DxDxDG motif to that of EF-hands had not been noted. The results obtained indicate that the EF-hand represents but one, among many, structural context for the DxDxDG-like Ca(2+)-binding loops. While the structural similarity of the binuclear calcium-binding sites in anthrax protective antigen and human thrombospondin suggests that they are homologous, evolutionary relationships for mononuclear sites are harder to discern. The possible scenarios for the evolution of DxDxDG motif-containing calcium-binding loops in a variety of non-homologous proteins suggested loop transplant as a mechanism perhaps responsible for much of the diversity in structural contexts of present day DxDxDG-type CaBPs. Additionally, while it can be shown that existence of a DxDxDG sequence is not enough to confer a conformation suitable for calcium binding, local convergent evolution may still have a role. The analysis presented here has consequences for the prediction of calcium binding from sequence alone.