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PubMed1999

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus. A triple-labeling immunofluorescence study.

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.

Animals↗
PubMed1999

Regional and cellular localisation of GABA(A) receptor subunits in the human basal ganglia: An autoradiographic and immunohistochemical study.

The regional and cellular localisation of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the human basal ganglia using receptor autoradiography and immunohistochemical staining for five GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), beta(2, 3), and gamma(2)) and other neurochemical markers. The results demonstrated that GABA(A) receptors in the striatum showed considerable subunit heterogeneity in their regional distribution and cellular localisation. High densities of GABA(A) receptors in the striosome compartment contained the alpha(2), alpha(3), beta(2, 3), and gamma(2) subunits, and lower densities of receptors in the matrix compartment contained the alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. Also, six different types of neurons were identified in the striatum on the basis of GABA(A) receptor subunit configuration, cellular and dendritic morphology, and chemical neuroanatomy. Three types of alpha(1) subunit immunoreactive neurons were identified: type 1, the most numerous (60%), were medium-sized aspiny neurons that were immunoreactive for parvalbumin and alpha(1), beta(2,3), and gamma(2) subunits; type 2 (38%) were medium-sized to large aspiny neurons immunoreactive for calretinin and alpha(1), alpha(3), beta(2,3), and gamma(2) subunits; and type 3 (2%) were large sparsely spiny neurons immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits. Type 4 neurons were calbindin-positive and immunoreactive for alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. The remaining neurons were immunoreactive for choline acetyltransferase (ChAT) and alpha(3) subunit (type 5) or were neuropeptide Y-positive with no GABA(A) receptor subunit immunoreactivity (type 6). The globus pallidus contained three types of neurons: types 1 and 2 were large neurons and were immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and for parvalbumin alone (type 1) or for both parvalbumin and calretinin (type 2); type 3 neurons were medium-sized and immunoreactive for calretinin and alpha(1), beta(2, 3), and gamma(2) subunits. These results show that the subunit composition of GABA(A) receptors displays considerable regional and cellular variation in the human striatum but are more homogeneous in the globus pallidus.

Acetylcholinesterase↗
PubMed2003

Abnormal neuronal expression of the calcium-binding proteins, parvalbumin and calbindin D-28k, in aged dogs.

Disturbances of the gamma-aminobutyric acid (GABA) neurotransmitter system have been implicated in chronic degenerative neurological disease. Cognitive dysfunction and neuron loss are features in older dogs. GABAergic neurons also show immunoreactivity for specific calcium-binding proteins. Immunohistochemistry was used to study the neuronal expression of calbindin D-28k and parvalbumin in different areas of the brain in 13 dogs, aged between 2 and 13.5 years. Calbindin expression was found only in the cerebellum. There were significant differences in the quantity and distribution of neurons expressing these proteins between geriatric and adult brains. Parvalbumin- and calbindin-expressing neurons are relatively sensitive to degeneration in the cerebellum of older dogs. Parvalbumin labelling was associated with dystrophic structures that are commonly associated with ageing.

Aging↗
PubMed1997

Molecular and physiological diversity of cortical nonpyramidal cells.

The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n = 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n = 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

Action Potentials↗
PubMed1996

Prolonged subordination stress increases Calbindin-D28k immunoreactivity in the rat hippocampal CA1 area.

Previously we observed that corticosteroids alter Calbindin-D28k immunoreactivity in the rat hippocampus. In the present study we investigated whether prolonged subordination stress, presumably producing elevated plasma corticosterone levels (1) altered the immunocytochemical distribution of the Ca(2+)-binding proteins Calbindin-D28k (CBir) and Parvalbumin (PVir) in the rat hippocampus, and (2) induced ongoing neurodegenerative changes using a silver impregnation method. Eight days of subordination stress reduced body weight, increased adrenal weight corrected for body weight and reduced thymus weight, indicating its effectiveness to produce a stressful situation. Stress increased CBir selectively in the CA1 pyramidal cell layer whereas PVir was not altered. Silver-impregnation revealed no ongoing neurodegenerative changes in any of the hippocampal subfields.

Animals↗
PubMed2000

Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats.

Phytoestrogen [plant estrogenic-like molecule(s)] research has grown rapidly in recent years due to their potential health benefits. However, little is known about phytoestrogen's effects on the CNS. Androgen metabolizing enzymes are known to regulate neuroendocrine functions and reproductive behaviors, while calcium-binding proteins are associated with protecting against neurodegenerative diseases. Therefore, we examined aromatase and 5alpha-reductase enzyme activities in the medial basal hypothalamic and preoptic area (mbh-poa) and characterized mbh-poa and amygdala (amy) calbindin and calretinin levels (via Western analysis) from animals fed a phytoestrogen-free (P-free) vs. a phytoestrogen-containing diet [(P-600); that had 600 microg/g of phytoestrogens]. After approximately 5 weeks on the diets, the male rats were killed at 105 days. P-600 plasma phytoestrogen levels were 78-fold higher than the P-free values and the mbh-poa phytoestrogen content was 8-fold higher than the P-free group, demonstrating the passage of phytoestrogens into brain. In general, brain aromatase or 5alpha-reductase activity levels were not significantly altered by the experimental diets. However, independent of brain site (i.e., mbh-poa or amy) the abundance of calbindin from male P-600 rats was significantly lower than P-free animals. Conversely, for calretinin there were no significant alterations in the mbh-poa tissue site, while in the amy a similar pattern of expression was seen to that of the calbindin results. These data suggest that consumption of phytoestrogens via a soy diet for a relatively short interval can significantly: (1) elevate plasma and brain phytoestrogens levels and (2) decrease brain calcium-binding proteins without altering brain androgen metabolizing enzymes.

Androgens↗
PubMed1996

Synaptophysin immunoreactivity and distributions of calcium-binding proteins highlight the functional organization of the rat's dorsal column nuclei.

The mammalian dorsal column nuclei (DCN) are principally composed of the cuneate (CN) and gracile (GN) nuclei. Data presented here support previously published anatomical and functional evidence that the longitudinal organization of the CN and GN reflect the complex role of the DCN in somatosensory processing. The CN is organized longitudinally into three parts. Within the middle portion of this nucleus, primary afferent projections and cuneothalamic cells are concentrated. Although traditional cytoarchitectonic analyses had failed to detect this tripartite organization in rats, we found evidence for it, with a functional middle region, extending approximately 0.2-0.9 mm caudal to the obex, characterized by precise somatotopy of primary afferent terminations and corresponding somatotopy of cytochrome oxidase (CO) blotches. Additional evidence is presented here consistent with a functionally distinct middle region within the rat's CN: (1) patches of dense synaptophysin (a synaptic-vesical-associated protein)-immunoreactivity (SYN-IR) are limited to the middle CN region, coincident with the dense CO blotches; (2) neurons immunoreactive for the calcium-binding proteins calbindin-D28 (CB), calretinin (CR) and parvalbumin (PV) are concentrated in the middle CN region. Furthermore, in adult rats subjected to perinatal forepaw removal, (1) the patterns of SYN-IR in the middle region of the CN are disrupted, as had previously been shown for the patterns of CO blotches; (2) in contrast, however, distributions of CN cells with PV-, CB- and CR-IR are unaffected. Evidence for a tripartite division in the GN is also presented, based on the distributions of cells with PV-, CB- and CR-IR.

Animals↗
PubMed2004

Effects of vitamin D compounds on renal and intestinal Ca2+ transport proteins in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice.

BACKGROUND: Vitamin D compounds are used clinically to control secondary hyperparathyroidism (SHPT) due to renal failure. Newer vitamin D compounds retain the suppressive action of 1,25(OH)(2)D(3) on the parathyroid glands and may have less Ca(2+)-mobilizing activity, offering potentially safer therapies. METHODS: This study investigated the effect of a single dose of compound (1,25(OH)(2)D(3), 1,24(OH)(2)D(2), or 1alpha(OH)D(2)) on renal and intestinal Ca(2+) transport proteins, including TRPV5 and TRPV6, and serum Ca(2+), in a novel SHPT model, the 25-OH-D(3)-1alpha-hydroxylase knockout mouse, which lacks endogenous 1,25(OH)(2)D(3) and is severely hypocalcemic. Animals were injected intraperitoneally with compound (100 ng/mouse). RESULTS: Serum levels of 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) peaked at four hours post-injection (pi), then declined rapidly. 1,25(OH)(2)D(2) generated from 1alpha(OH)D(2) peaked at 12 hours pi and then remained stable. Serum Ca(2+) was increased to near-normal within four hours by 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2), and within 12 hours by 1alpha(OH)D(2). 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) up-regulated duodenal TRPV5 and TRPV6 mRNA to a similar degree within four hours; mRNA levels decreased by 12 hours after 1,24(OH)(2)D(2) treatment, and by 24 hours after 1,25(OH)(2)D(3) treatment. 1,25(OH)(2)D(3) increased kidney levels of TRPV5, calbindin-D(28K), and calbindin-D(9K) mRNA within four hours; 1,24(OH)(2)D(2) did not change kidney TRPV5 levels and modestly increased calbindin D(9K) by 48 hours. 1alpha(OH)D(2) produced later-onset effects, increasing duodenal TRPV6 and calbindin-D(9K) mRNA levels by 12 hours and TRPV5 by 48 hours. CONCLUSION: In kidney, 1alpha(OH)D(2) increased TRPV5, calbindin-D(28K), and calbindin-D(9K) mRNA levels by 12 hours. This study indicates that Ca(2+) transport proteins, including TRPV5 and TRPV6, are differentially up-regulated by vitamin D compounds.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1992

Identification of neuron subpopulations in the rat vestibular ganglion by calbindin-D 28K, calretinin and neurofilament proteins immunoreactivity.

Immunocytochemical and morphometric analyses were combined to demonstrate the presence of neuron subpopulations in the rat vestibular ganglion. Monoclonal antibodies reacting with neurofilament proteins (NF), calbindin-D 28K (CaBP) and calretinin (CaR) were used. Three subpopulations were identified: (1) CaBP- and CaR-positive neurons were the largest neurons (16%) and they were also highly NF-immunoreactive; (2) exclusively NF-positive neurons; (3) unlabelled neurons, representing about two-thirds of the population.

Animals↗
PubMed2004

Gene expression profiles in rat intestine identify pathways for 1,25-dihydroxyvitamin D(3) stimulated calcium absorption and clarify its immunomodulatory properties.

Microarray technology has been used to discover 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) induced gene expression changes in rat small intestine in vivo. Here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to 1,25-(OH)(2)D(3). Vitamin D deficient rats were intrajugularly given vehicle or vehicle containing 730 ng of 1,25-(OH)(2)D(3)/kg of body weight. Intestinal mRNA was harvested from duodenal mucosa at 15 min, 1, 3, and 6 h post-injection and studied by Affymetrix microarrays. Genes significantly affected by 1,25-(OH)(2)D(3) were confirmed by quantitative RT-PCR with remarkable agreement. The most strongly affected gene in intestine was CYP24 with 97-fold increase at 6 h post-1,25-(OH)(2)D(3) treatment. Intestinal calcium absorption genes: TRPV5, TRPV6, calbindin D(9k), and Ca(2+) dependent ATPase all were up-regulated in response to 1,25-(OH)(2)D(3), supporting the currently accepted mechanism of 1,25-(OH)(2)D(3) induced transcellular calcium transport. However, a 1,25-(OH)(2)D(3) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium ATPase, claudin 3, aquaporin 8, cadherin 17, and RhoA suggests a vitamin D regulation of tight junction permeability and paracellular calcium transport. Several other genes related to the immune system and angiogenesis whose expression was changed in response to 1,25-(OH)(2)D(3) provided evidence for an immunomodulatory and anti-angiogenic role of 1,25-(OH)(2)D(3).

Administration, Oral↗
PubMed2003

Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k.

UNLABELLED: This study show for the first time that calbindin-D28k can prevent glucocorticoid-induced bone cell death. The anti-apoptotic effect of calbindin-D28k involves inhibition of glucocorticoid induced caspase 3 activation as well as ERK activation. INTRODUCTION: Recent studies have indicated that deleterious effects of glucocorticoids on bone involve increased apoptosis of osteocytes and osteoblasts. Because the calcium-binding protein calbindin-D28k has been reported to be anti-apoptotic in different cell types and in response to a variety of insults, we investigated whether calbindin-D28k could protect against glucocorticoid-induced cell death in bone cells. MATERIALS AND METHODS: Apoptosis was induced by addition of dexamethasone (dex; 10-6 M) for 6 h to MLO-Y4 osteocytic cells as well as to osteoblastic cells. Apoptosis percentage was determined by examining the nuclear morphology of transfected cells. Caspase 3 activity was evaluated in bone cells and in vitro. SELDI mass spectrometry (MS) was used to examine calbindin-D28k-caspase 3 interaction. Phosphorylation of calbindin-D28k was examined by 32P incorporation as well as by MALDI-TOF MS. ERK activation was determined by Western blot. RESULTS: The pro-apoptotic effect of dex in MLO-Y4 cells was completely inhibited in cells transfected with calbindin-D28k cDNA (5.6% apoptosis in calbindin-D28k transfected cells compared with 16.2% apoptosis in vector-transfected cells, p < 0.05). Similar results were observed in osteoblastic cells. We found that dex-induced apoptosis in bone cells was accompanied by an increase in caspase 3 activity. This increase in caspase 3 activity was inhibited in the presence of calbindin-D28k. In vitro assays indicated a concentration-dependent inhibition of caspase 3 by calbindin-D28k (Ki = 0.22 microM). Calbindin-D28k was found to inhibit caspase 3 specifically because the activity of other caspases was unaffected by calbindin-D28k. The anti-apoptotic effect of calbindin-D28k in response to dex was also reproducibly associated with an increase in the phosphorylation of ERK 1 and 2, suggesting that calbindin-D28k affects more than one signal in the glucocorticoid-induced apoptotic pathway. CONCLUSION: Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.

Animals↗
PubMed2004

Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats.

UNLABELLED: The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)(2)D(3) increased VDR and calbindins in controls but not GHS rats. VDR half-life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats. INTRODUCTION: Genetic hypercalciuric stone-forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D-responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known. MATERIALS AND METHODS: GHS and non-stone-forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0-8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots. RESULTS AND CONCLUSIONS: Duodenal VDR protein by Western blot was increased 2-fold in GHS versus NC rats (633 +/- 62 versus 388 +/- 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half-life was prolonged in GHS rats (2.59 +/- 0.2 versus 1.81 +/- 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half-life in NC rats to that of untreated GHS rats (2.59 +/- 0.2 versus 2.83 +/- 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half-life increases VDR tissue levels and mediates increased VDR-regulated genes that result in hypercalciuria through actions on vitamin D-regulated calcium transport in intestine, bone, and kidney.

Animals↗
PubMed1996

Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: I. Cell morphology and morphometrics.

This paper provides a comprehensive morphological description of local circuit neurons in the medial prefrontal cortex (mPFC: areas 24a, 24b, 24c, 25 and 32) of the monkey. Cortical interneurons were identified immunocytochemically by the expression of the calcium binding proteins calretinin (CR), parvalbumin (PV) and calbindin D-28k (CB). Interneurons were also identified using GABA immunocytochemistry. The areal and laminar distributions of CR, PV, and CB cells were consistent across mPFC; their morphological characteristics identified them as local circuit neurons. Throughout layers 2-6: CR immunoreactivity labelled double bouquet and bipolar neurons, PV was localised in large and small basket neurons and in chandelier (axoaxonic) cells, while CB immunoreactivity was present in double bouquet, Martinotti, and neurogliaform neurons. In addition, some cells in layer 1 (including Cajal-Retzius neurons) were CR immunoreactive. Calbindin immunoreactivity also labelled a population of large nonpyramidal neurons deep in the cortex. Other types of CR, PV and CB cells were also immunolabelled. A small population of layer 3 pyramidal cells was weakly CB immunoreactive. Peak cell densities occurred in layer 2/upper layer 3 for CR+ neurons and in upper to midlayer 3 for CB+ cells. PV+ neuron density peaked in midcortex. These observations support and extend a similar study of monkey prefrontal cortex (Condé et al. [1994] J. Comp. Neurol. 341:95-116). The morphologies and combined cortical depth distributions of CR+, PV+, and CB+ neurons were similar to GABA-immunolabelled cells. Local circuit neurons in mPFC displaying NADPH diaphorase activity composed less than 0.25% of the total neuron population, and were distributed in two horizontal strata, in mid- to lower layer 3 and in lower layer 5/upper layer 6. CR, PV and CB immunoreactivity was colocalised in NADPH diaphorase-reactive neurons. The interrelationships between CR+, PV+ and CB+ neurons were investigated using dual immunocytochemistry. CR+ puncta were found to be closely associated with the cell bodies and proximal processes of PV+ neurons, whereas CR+ puncta were located more distally over processes from CB+ cells. Additionally, PV+ puncta were found closely apposed to PV+ somata and processes and CR+ puncta abutted against CR+ cell bodies. The companion paper (Gabbott and Bacon [1996] J. Comp. Neurol.) presents quantitative data regarding the areal and laminar distributions of the identified cell classes in mPFC. Such data provide a realistic structural framework with which to investigate neuronal operations in monkey mPFC.

Animals↗
PubMed1994

Deafferentation removes calretinin immunopositive terminals, but does not induce degeneration of calbindin D-28k and parvalbumin expressing neurons in the hippocampus of adult rats.

Unilateral combined transections of the fimbriafornix and angular bundle in adult Fischer 344 rats were used to study the effects of deafferentation on hippocampal expression of calretinin, calbindin D-28k, and parvalbumin. Reflecting the widespread degeneration of synaptic contacts, immunostaining for glial fibrillary acidic protein 6 days after the lesions was increased in lacunosum-molecular and oriens layers of CA1, 2, and 3 in ipsi- and contralateral hippocampus and in the ipsilateral dentate gyrus outer molecular layer. At 21 days the immunoreactivity had decreased to control levels except for a still slightly increased signal in the oriens layer of CA1-3. At 6 and 21 days after the combined lesions the numbers of hippocampal neurons containing calretinin, parvalbumin, and calbindin D-28k was unaltered. The combined lesions abolished calretinin containing terminals in the dentate gyrus inner molecular layer on the deafferentated side. This could be reproduced by single unilateral fimbria-fornix transections, suggesting that the axons of these calretinin positive terminals project to the hippocampus through the fimbria-fornix. The most likely origin of the calretinin positive terminals are neurons in the supramammillary hypothalamic nucleus. Our findings demonstrate that the extensive lesion-induced synaptic rearrangements in the adult hippocampus do not induce degeneration of hippocampal neurons expressing calretinin, calbindin D-28k, and parvalbumin, but do remove calretinin containing terminals which reach their targets in the hippocampus through the fimbria-fornix.

Animals↗
PubMed2001

Structure of rat parvalbumin with deleted AB domain: implications for the evolution of EF hand calcium-binding proteins and possible physiological relevance.

Among the EF-hand Ca(2+)-binding proteins, parvalbumin (PV) and calbindin D9k (CaB) have the function of Ca(2+) buffers. They evolved from an ancestor protein through two phylogenetic pathways, keeping one pair of EF-hands. They differ by the extra helix-loop-helix (AB domain) found in PV and by the linker between the binding sites. To investigate whether the deletion of AB in PV restores a CaB-like structure, we prepared and solved the structure of the truncated rat PV (PVratDelta37) by X-ray and NMR. PVratDelta37 keeps the PV fold, but is more compact, having a well-structured linker, which differs remarkably from CaB. PvratDelta37 has no stable apo-form, has lower affinity for Ca(2+) than full-length PV, and does not bind Mg(2+), in contrast to CaB. Structural differences of the hydrophobic core are partially responsible for lowering the calcium-binding affinity of the truncated protein. It can be concluded that the AB domain, like the linker of CaB, plays a role in structural stabilization. The AB domain of PV protects the hydrophobic core, and is required to maintain high affinity for divalent cation binding. Therefore, the AB domain possibly modulates PV buffer function.

Amino Acid Sequence↗
PubMed2002

NECABs: a family of neuronal Ca(2+)-binding proteins with an unusual domain structure and a restricted expression pattern.

Ca(2+)-signalling plays a major role in regulating all aspects of neuronal function. Different types of neurons exhibit characteristic differences in the responses to Ca(2+)-signals. Correlating with differences in Ca(2+)-response are expression patterns of Ca(2+)-binding proteins that often serve as markers for various types of neurons. For example, in the cerebral cortex the EF-hand Ca(2+)-binding proteins parvalbumin and calbindin are primarily expressed in inhibitory interneurons where they influence Ca(2+)-dependent responses. We have now identified a new family of proteins called NECABs (neuronal Ca(2+)-binding proteins). NECABs contain an N-terminal EF-hand domain that binds Ca(2+), but different from many other neuronal EF-hand Ca(2+)-binding proteins, only a single EF-hand domain is present. At the C-terminus, NECABs include a DUF176 motif, a bacterial domain of unknown function that was previously not observed in eukaryotes. In rat at least three closely related NECAB genes are expressed either primarily in brain (NECABs 1 and 2) or in brain and muscle (NECAB 3). Immunocytochemistry revealed that NECAB 1 is restricted to subsets of neurons. In cerebral cortex, NECAB 1 is highly and uniformly expressed only in layer 4 pyramidal neurons, whereas in hippocampus only inhibitory interneurons and CA2 pyramidal cells contain NECAB 1. In these neurons, NECAB 1 fills the entire cytoplasm similar to other EF-hand Ca(2+)-binding proteins, and is not concentrated in any particular subcellular compartment. We suggest that NECABs represent a novel family of regulatory Ca(2+)-binding proteins with an unusual domain structure and a limited expression in a subclass of neurons.

Amino Acid Sequence↗
PubMed1991

Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells.

Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin-Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation.

Adenosine Triphosphate↗
PubMed1996

Midbrain dopaminergic neurons in the mouse: co-localization with Calbindin-D28K and calretinin.

The calcium-binding proteins Calbindin-D28k and calretinin are co-localized with dopamine in some of the midbrain dopaminergic neurons in the rat and monkey; the present study sought to examine the pattern of co-localization in the mouse. Double immunofluorescence staining procedures were used for tyrosine hydroxylase (a dopaminergic cell marker) and Calbindin-D28k or calretinin. Midbrain dopaminergic neurons were examined at four rostrocaudal levels, and the percentage of cells that contained both tyrosine hydroxylase and either of the two calcium-binding proteins was determined in nucleus A8 (retrorubral field), nucleus A9 (substantia nigra pars compacta, pars reticulata and pars lateralis) and nucleus A10 (nucleus paranigralis, ventral tegmental area, interfascicular nucleus, central linear nucleus). The two calcium-binding proteins were distributed similarly in midbrain dopaminergic neurons in the several nuclear groups that comprise nuclei A8, A9 and A10. The calcium-binding proteins were found in the majority (50-100%) of nucleus A10 neurons, whereas in nuclei A8 and A9 (except for the substantia nigra pars lateralis) less than 40% of the cells contained either calcium-binding protein. The pattern of co-localization in the mouse is similar to that reported for the rat and monkey. The calcium-binding proteins mark the population of midbrain dopaminergic neurons that are less vulnerable to degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

Animals↗