Determination of the membrane topology of the small EF-hand Ca2+-sensing proteins CaBP7 and CaBP8

The CaBPs represent a subfamily of small EF-hand containing calcium (Ca(2+))-sensing proteins related to calmodulin that regulate key ion channels in the mammalian nervous system. In a recent bioinformatic analyses we determined that CaBP7 and CaBP8 form an evolutionarily distinct branch within the CaBPs (also known as the calneurons) a finding that is consistent with earlier observations characterising a putative C-terminal transmembrane (TM) spanning helix in each of these proteins which is essential for their sub-cellular targeting to the Golgi apparatus and constitutive secretory vesicles. The C-terminal position of the predicted TM-helix suggests that CaBP7 and CaBP8 could be processed in a manner analogous to tail-anchored integral membrane proteins which exhibit the ability to insert across membranes post-translationally. In this study we have investigated the topology of CaBP7 and CaBP8 within cellular membranes through a combination of trypsin protection and epitope accessibility analyses. Our results indicate that the TM-helices of CaBP7 and CaBP8 insert fully across membranes such that their extreme C-termini are luminal. The observed type-II membrane topology is consistent with processing of CaBP7 and CaBP8 as true tail-anchored proteins. This targeting mechanism is distinct from any other calmodulin related Ca(2+)-sensor and conceivably underpins unique physiological functions of these proteins.     

CUDASW++2.0: enhanced Smith-Waterman protein database search on CUDA-enabled GPUs based on SIMT and virtualized SIMD abstractions

Background

Ca²⁺-binding proteins are important for the transduction of Ca²⁺ signals into physiological outcomes. As in calmodulin many of the Ca²⁺-binding proteins bind Ca²⁺ through EF-hand motifs. Amongst the large number of EF-hand containing Ca²⁺-binding proteins are a subfamily expressed in neurons and retinal photoreceptors known as the CaBPs and the related calneuron proteins. These were suggested to be vertebrate specific but exactly which family members are expressed outside of mammalian species had not been examined.

Findings

We have carried out a bioinformatic analysis to determine when members of this family arose and the conserved aspects of the protein family. Sequences of human members of the family obtained from GenBank were used in Blast searches to identify corresponding proteins encoded in other species using searches of non-redundant proteins, genome sequences and mRNA sequences. Sequences were aligned and compared using ClustalW. Some families of Ca²⁺-binding proteins are known to show a progressive expansion in gene number as organisms increase in complexity. In contrast, the results for CaBPs and calneurons showed that a full complement of CaBPs and calneurons are present in the teleost fish Danio rerio and possibly in cartilaginous fish. These findings suggest that the entire family of genes may have arisen at the same time during vertebrate evolution. Certain members of the family (for example the short form of CaBP1 and calneuron 1) are highly conserved suggesting essential functional roles.

Conclusions

The findings support the designation of the calneurons as a distinct sub-family. While the gene number for CaBPs/calneurons does not increase, a distinctive evolutionary change in these proteins in vertebrates has been an increase in the number of splice variants present in mammals.     

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7: A REGULATOR OF GOLGI TRAFFICKING*

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca²⁺ binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca²⁺ but not Mg²⁺ and undergoes significant conformational changes in both secondary and tertiary structure upon Ca²⁺ binding. The Ca²⁺-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca²⁺-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.     

Decalmodulation of Cav1 channels by CaBPs

Ca²⁺-dependent inactivation (CDI) is a negative feedback regulation of voltage-gated Ca_v1 and Ca_v2 channels that is mediated by the Ca²⁺ sensing protein, calmodulin (CaM), binding to the pore-forming Ca_v α₁ subunit. David Yue and his colleagues made seminal contributions to our understanding of this process, as well as factors that regulate CDI. Important in this regard are members of a family of Ca²⁺ binding proteins (CaBPs) that are related to calmodulin. CaBPs are expressed mainly in neural tissues and can antagonize CaM-dependent CDI for Ca_v1 L-type channels. This review will focus on the roles of CaBPs as Ca_v1-interacting proteins, and the significance of these interactions for vision, hearing, and neuronal Ca²⁺ signaling events.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca²⁺-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Here we present NMR structures of CaBP1 in both Mg²⁺-bound and Ca²⁺-bound states and their structural interaction with InsP₃Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg²⁺ bound at EF1. The C-domain binds Ca²⁺ at EF3 and EF4, and exhibits a Ca²⁺-induced closed to open transition like that of CaM. The Ca²⁺-bound C-domain contains exposed hydrophobic residues (Leu¹³², His¹³⁴, Ile¹⁴¹, Ile¹⁴⁴, and Val¹⁴⁸) that may account for selective binding to InsP₃Rs. Isothermal titration calorimetry analysis reveals a Ca²⁺-induced binding of the CaBP1 C-domain to the N-terminal region of InsP₃R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP₃Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP₃ binding to the receptor. We propose that CaBP1 may regulate Ca²⁺-dependent activity of InsP₃Rs by promoting structural contacts between the suppressor and core domains.Calcium ion (Ca²⁺) in the cell functions as an important messenger that controls neurotransmitter release, gene expression, muscle contraction, apoptosis, and disease processes (1). Receptor stimulation in neurons promotes large increases in intracellular Ca²⁺ levels controlled by Ca²⁺ release from intracellular stores through InsP₃Rs (2). The neuronal type-1 receptor (InsP₃R1)² is positively and negatively regulated by cytosolic Ca²⁺ (3-6), important for the generation of repetitive Ca²⁺ transients known as Ca²⁺ spikes and waves (1). Ca²⁺-dependent activation of InsP₃R1 contributes to the fast rising phase of Ca²⁺ signaling known as Ca²⁺-induced Ca²⁺ release (7). Ca²⁺-induced inhibition of InsP₃R1, triggered at higher cytosolic Ca²⁺ levels, coordinates the temporal decay of Ca²⁺ transients (6). The mechanism of Ca²⁺-dependent regulation of InsP₃Rs is complex (8, 9), and involves direct Ca²⁺ binding sites (5, 10) as well as remote sensing by extrinsic Ca²⁺-binding proteins such as CaM (11, 12), CaBP1 (13, 14), CIB1 (15), and NCS-1 (16).Neuronal Ca²⁺-binding proteins (CaBP1-5 (17)) represent a new sub-branch of the CaM superfamily (18) that regulate various Ca²⁺ channel targets. Multiple splice variants and isoforms of CaBPs are localized in different neuronal cell types (19-21) and perform specialized roles in signal transduction. CaBP1, also termed caldendrin (22), has been shown to modulate the Ca²⁺-sensitive activity of InsP₃Rs (13, 14). CaBP1 also regulates P/Q-type voltage-gated Ca²⁺ channels (23), L-type channels (24), and the transient receptor potential channel, TRPC5 (25). CaBP4 regulates Ca²⁺-dependent inhibition of L-type channels in the retina and may be genetically linked to retinal degeneration (26). Thus, the CaBP proteins are receiving increased attention as a family of Ca²⁺ sensors that control a variety of Ca²⁺ channel targets implicated in neuronal degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in CaM and troponin C (18) (Fig. 1). By analogy to CaM (27), the four EF-hands are grouped into two domains connected by a central linker that is four residues longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain non-conserved amino acids within the N-terminal region that may confer target specificity. Another distinguishing property of CaBPs is that the second EF-hand lacks critical residues required for high affinity Ca²⁺ binding (17). CaBP1 binds Ca²⁺ only at EF3 and EF4, whereas it binds Mg²⁺ at EF1 that may serve a functional role (28). Indeed, changes in cytosolic Mg²⁺ levels have been detected in cortical neurons after treatment with neurotransmitter (29). Other neuronal Ca²⁺-binding proteins such as DREAM (30), CIB1 (31), and NCS-1 (32) also bind Mg²⁺ and exhibit Mg²⁺-induced physiological effects. Mg²⁺ binding in each of these proteins helps stabilize their Ca²⁺-free state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary structural elements (α-helices and β-strands) were derived from NMR analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted green, red, cyan, and yellow. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Non-conserved residues in the hydrophobic patch are colored red.Despite extensive studies on CaBP1, little is known about its structure and target binding properties, and regulation of InsP₃Rs by CaBP1 is somewhat controversial and not well understood. Here, we present the NMR solution structures of both Mg²⁺-bound and Ca²⁺-bound conformational states of CaBP1 and their structural interactions with InsP₃R1. These CaBP1 structures reveal important Ca²⁺-induced structural changes that control its binding to InsP₃R1. Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca²⁺-induced binding to the N-terminal cytosolic region of InsP₃R1. We propose that CaBP1 may regulate Ca²⁺-dependent channel activity in InsP₃Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca²⁺-dependent channel gating (8, 33, 34).     

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

CaBP4 modulates Ca²⁺-dependent activity of L-type voltage-gated Ca²⁺ channels (Cav1.4) in retinal photoreceptor cells. Mg²⁺ binds to the first and third EF-hands (EF1 and EF3), and Ca²⁺ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg²⁺-bound and Ca²⁺-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1–99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg²⁺ or Ca²⁺ bound at EF1. The C-lobe binds Ca²⁺ at EF3 and EF4 and exhibits a Ca²⁺-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca²⁺-bound CaBP4 (Phe¹³⁷, Glu¹⁶⁸, Leu²⁰⁷, Phe²¹⁴, Met²⁵¹, Phe²⁶⁴, and Leu²⁶⁸) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca²⁺-dependent inactivation domain.     

Complex Regulation of Voltage-dependent Activation and Inactivation Properties of Retinal Voltage-gated Cav1.4 L-type Ca2+ Channels by Ca2+-binding Protein 4 (CaBP4)

Cav1.4 L-type Ca²⁺ channels are crucial for synaptic transmission in retinal photoreceptors and bipolar neurons. Recent studies suggest that the activity of this channel is regulated by the Ca²⁺-binding protein 4 (CaBP4). In the present study, we explored this issue by examining functional effects of CaBP4 on heterologously expressed Cav1.4. We show that CaBP4 dramatically increases Cav1.4 channel availability. This effect crucially depends on the presence of the C-terminal ICDI (inhibitor of Ca²⁺-dependent inactivation) domain of Cav1.4 and is absent in a Cav1.4 mutant lacking the ICDI. Using FRET experiments, we demonstrate that CaBP4 interacts with the IQ motif of Cav1.4 and that it interferes with the binding of the ICDI domain. Based on these findings, we suggest that CaBP4 increases Cav1.4 channel availability by relieving the inhibitory effects of the ICDI domain on voltage-dependent Cav1.4 channel gating. We also functionally characterized two CaBP4 mutants that are associated with a congenital variant of human night blindness and other closely related nonstationary retinal diseases. Although both mutants interact with Cav1.4 channels, the functional effects of CaBP4 mutants are only partially preserved, leading to a reduction of Cav1.4 channel availability and loss of function. In conclusion, our study sheds new light on the functional interaction between CaBP4 and Cav1.4. Moreover, it provides insights into the mechanism by which CaBP4 mutants lead to loss of Cav1.4 function and to retinal disease.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

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).     

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).     

Alteration of CaBP Expression Pattern in the Nucleus Magnocellularis following Unilateral Cochlear Ablation in Adult Zebra Finches

Songbirds have the rare ability of auditory-vocal learning and maintenance. Up to now, the organization and function of the nucleus magnocellularis (NM), the first relay of the avian ascending auditory pathway is largely based on studies in non-vocal learning species, such as chickens and owls. To investigate whether NM exhibits different histochemical properties associated with auditory processing in songbirds, we examined the expression patterns of three calcium-binding proteins (CaBPs), including calretinin (CR), parvalbumin (PV) and calbindin-D28k (CB), and their relations to auditory inputs in NM in adult zebra finches. We found enriched and co-localized immunostaining of CR, PV and CB in the majority of NM neurons, without neuronal population preference. Furthermore, they were sensitive to adult deafferentation with differential plasticity patterns. After unilateral cochlear removal, CR staining in the ipsilateral NM decreased appreciably at 3 days after surgery, and continued to decline thereafter. PV staining showed down-regulation first at 3 days, but subsequently recovered slightly. CB staining did not significantly decrease until 7 days after surgery. Our findings suggest that the three CaBPs might play distinct roles in association with auditory processing in zebra finches. These results are in contrast to the findings in the NM of chickens where CR is the predominant CaBP and deafferentation had no apparent effect on its expression. Further extended studies in other avian species are required to establish whether the difference in CaBP patterns in NM is functionally related to the different auditory-vocal behaviors.     

Isolation of two isoforms of a 21,000-dalton Ca2+-binding protein of bovine brain

Using Ca2+-dependent hydrophobic interaction chromatography we have identified a novel bovine brain Ca2+-binding protein (CaBP) composed of 21 kDa and 23 kDa polypeptides. This calciprotein was further purified by heat-treatment in the presence of Ca2+ and ion-exchange chromatography. The isolated protein exhibits a number of properties in common with proteins belonging to the calmodulin family of CaBPs, including a Ca2+-dependent electrophoretic mobility shift on SDS-polyacrylamide gel electrophoresis, retention of the ability to bind 45Ca2+ after electrophoresis and Western blotting, and a high content of acidic amino acids. We have recently isolated and characterized a 21 kDa CaBP from bovine brain and conclude that the 21 kDa and 21/23 kDa CaBPs are isoforms since they have very similar U.V. absorption spectra and amino acid compositions, and polyclonal antibodies raised in rabbits against the 21 kDa CaBP cross-react to an identical degree with the 21/23 kDa CaBP as determined by the competitive enzyme-linked immunosorbent assay (ELISA). Both proteins contain carbohydrate, but they differ in the degree of glycosylation. Tissue distribution studies indicate the presence of both 21 kDa and 23 kDa Ca2+-binding polypeptides in bovine trachea, aorta, kidney, skeletal muscle and cardiac muscle, and chicken gizzard smooth muscle.     

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

Structural and functional diversity of Entamoeba histolytica calcium-binding proteins

Entamoeba histolytica (E. histolytica) is an etiological agent of human amoebic colitis, and it causes a high level of morbidity and mortality worldwide, particularly in developing countries. Ca²⁺ plays a pivotal role in amoebic pathogenesis, and Ca²⁺-binding proteins (CaBPs) of E. histolytica appear to be a major determinant in this process. E. histolytica has 27-EF-hand containing CaBPs, suggesting that this organism has complex Ca²⁺ signaling cascade. E. histolytica CaBPs share (29–47%) sequence identity with ubiquitous Ca²⁺-binding protein calmodulin (CaM); however, they do not show any significant structural similarity, indicating lack of a typical CaM in this organism. Structurally, these CaBPs are very diverse among themselves, and perhaps such diversity allows them to recognize different cellular targets, thereby enabling them to perform a range of cellular functions. The presence of such varied signaling molecules helps parasites to invade host cells and advance in disease progression. In the past two decades, tremendous progress has been made in understanding the structure of E. histolytica CaBPs by using the X-ray or NMR method. To gain greater insight into the structural and functional diversity of these amoebic CaBPs, we analyzed and compiled all the available literature. Most of the CaBPs has about 150 amino acids with 4-EF hand or EF-hand-like sequences, similar to CaM. In a few cases, all the EF-hand motifs are not capable of binding Ca²⁺, suggesting them to be pseudo EF-hand motifs. The CaBPs perform diverse cellular signaling that includes cytoskeleton remodeling, phagocytosis, cell proliferation, migration of trophozoites, and GTPase activity. Overall, the structural and functional diversity of E. histolytica CaBPs compiled here may offer a basis to develop an efficient drug to counter its pathogenesis.     

Potassium-dependent sodium-calcium exchanger (NCKX) isoforms and neuronal function

K⁺-dependent Na⁺/Ca²⁺-exchangers (NCKX) are a relatively recently described five-member gene family of transporters which play a quantitatively significant role in neuronal Ca²⁺ transport. In this review we highlight the important individual contributions these transporters make to cellular Ca²⁺ homeostasis and neuronal function. Notably, different members of the family make distinct, non-redundant, contributions to critical behavioural pathways. In particular, NCKX proteins regulate the kinetics, termination and adaptation of Ca²⁺ signals in sensory transduction neurons in the olfactory and visual systems. Similar contributions to shaping the spatial and temporal features of Ca²⁺ signals in neurons at other key brain locations have important consequences for the circuitry influencing control of satiety, for experience-dependent motor learning and spatial working memory retention, as well as in the protection of neurons in the face of toxic stimuli. NCKX proteins are also key contributors to a variety of events in other tissues. The connection between NCKX isoform function and human phenotype and disease is an emerging area, and we anticipate that future research will reveal rich new details in the coming years.     

Calcium-binding proteins: intracellular sensors from the calmodulin superfamily.

In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.     

Calcium-binding protein 1 is an inhibitor of agonist-evoked, inositol 1,4,5-trisphosphate-mediated calcium signaling

Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.     

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Calcium signaling in neurons as in other cell types mediates changes in gene expression, cell growth, development, survival, and cell death. However, neuronal Ca²⁺ signaling processes have become adapted to modulate the function of other important pathways including axon outgrowth and changes in synaptic strength. Ca²⁺ plays a key role as the trigger for fast neurotransmitter release. The ubiquitous Ca²⁺ sensor calmodulin is involved in various aspects of neuronal regulation. The mechanisms by which changes in intracellular Ca²⁺ concentration in neurons can bring about such diverse responses has, however, become a topic of widespread interest that has recently focused on the roles of specialized neuronal Ca²⁺ sensors. In this article, we summarize synaptotagmins in neurotransmitter release, the neuronal roles of calmodulin, and the functional significance of the NCS and the CaBP/calneuron protein families of neuronal Ca²⁺ sensors.Calcium signaling in many cell types can mediate changes in gene expression, cell growth, development, survival, and cell death. However, neuronal calcium signaling processes have become adapted to modulate the function of important pathways in the brain, including neuronal survival, axon outgrowth, and changes in synaptic strength. Changes in the concentration of intracellular free Ca²⁺ ([Ca²⁺]i) are essential for the transmission of information through the nervous system as the trigger for neurotransmitter release at synapses. In addition, alterations in [Ca²⁺]i can lead to a wide range of different physiological changes that can modify neuronal functions over time scales of milliseconds through tens of minutes to days or longer (Berridge 1998). Many of these processes have been shown to be dependent upon the particular route of Ca²⁺ entry into the cell. It has long been known that the physiological outcome from a change in [Ca²⁺]i depends on its location, amplitude, and duration. The importance of location becomes even more pronounced in neurons because of their complex and extended morphologies. [Ca²⁺]i also regulates neuronal development and neuronal survival (Spitzer 2006). In addition, modifications to Ca²⁺ signaling pathways have been suggested to underlie various neuropathological disorders (Braunewell 2005; Berridge 2010).Highly localized Ca²⁺ elevations (Augustine et al. 2003) formed following Ca²⁺ entry though voltage-gated Ca²⁺ channels (VGCCs) lead to synaptic vesicle fusion with the presynaptic membrane and thereby allow neurotransmitter release within less than a millisecond. Differently localized and timed Ca²⁺ signals can, for example, result in changes to the properties of the VGCCs (Catterall and Few 2008) or lead to changes in gene expression (Bito et al. 1997). Postsynaptic Ca²⁺ signals arising from activation of NMDA receptors give rise to two important processes in synaptic plasticity, long term potentiation (LTP) and long term depression (LTD). LTP and LTD are examples of the way synaptic transmission can change synaptic efficacy and are thought to be important in modulating learning and memory. Importantly, the Ca²⁺ signals that bring about either LTP or LTD differ only in their timing and duration. LTP is triggered by Ca²⁺ signals on the micromolar scale for shorter durations, whereas LTD is triggered by changes in [Ca²⁺]i on the nanomolar scale for longer durations (Yang et al. 1999). Specific Ca²⁺ signals are likely to be decoded by different Ca²⁺ sensor proteins. These are proteins that undergo a conformational change on Ca²⁺ binding and then interact with and regulate various target proteins. Among those Ca²⁺ sensors that are important for neuronal function are the synaptotagmins that control neurotransmitter release (Chapman 2008), the ubiquitous EF-hand containing sensor calmodulin that has many neuronal roles, and the more recently discovered neuronal EF-hand containing proteins, including the neuronal calcium sensor (NCS) protein (Burgoyne 2007) and the calcium-binding protein (CaBP)/calneuron (Haeseleer et al. 2002) families. We will briefly review synaptotagmins and the neuronal functions of calmodulin but concentrate on the NCS and CaBP families of Ca²⁺ sensors.