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PubMed2003

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k.

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.

Amides↗
PubMedDate not supplied

Probing fragment complementation by rigid-body docking: in silico reconstitution of calbindin D9k.

Fragment complementation is gaining an increasing impact as a nonperturbing method to probe noncovalent interactions within protein supersecondary structures. In this study, the fast Fourier transform rigid-body docking algorithm ZDOCK has been employed for in silico reconstitution of the calcium binding protein calbindin D9k, from its two EF-hands subdomains, namely, EF1 (residues 1-43) and EF2 (residues 44-75). The EF1 fragment has been used both in its wild type and in nine mutant forms, in line with in vitro experiments. Consistent with in vitro data, ZDOCK reconstituted the proper fold of wild-type and mutated calbindin, locating the nativelike structures (i.e., holding a root-mean-square deviation < 1 A with respect to the X-ray structure) among the first 10 top-scored solutions out of 4000. Moreover, the three independent in silico reconstitutions of wild-type calbindin ranked a nativelike structure at the top of the output list, that is, the best scored one. The algorithm has been also successfully challenged in reconstituting the EF2 homodimer from two identical copies of the monomer. Furthermore, quantitative models consisting of linear correlations between thermodynamic data and ZDOCK scores were built, providing a tested tool for very fast in silico predictions of the free energy of association of protein-protein complexes solved at the atomic level and known to not undergo significant conformational changes upon binding.

Calbindins↗
PubMedDate not supplied

Calbindin immunoreactivity alternates with cytochrome c-oxidase-rich zones in some layers of the primate visual cortex.

Calcium ions have a pivotal role in many neuronal activities, but little is known about their involvement in the cortical processing of visual information. Using immunohistochemical methods, we have now detected a calcium-binding protein, calbindin-D-28K, which may confer on certain compartments of cortical area 17 the ability to modulate Ca2+ metabolism. Thus, calbindin occurs in the primate striate cortex in a pattern almost complementary to that displaying strong cytochrome c-oxidase activity. From this and other observations, we deduce that the distribution of calbindin-immunoreactive sites corresponds mainly to extra-geniculocortical connections of the primary visual cortex. This implies that the geniculocortical and extra-geniculocortical compartments of area 17 differ in an intracellular system for Ca2+ homeostasis.

Animals↗
PubMed2000

Comparison of localization of the neurokinin 1 receptor and nitric oxide synthase with calbindin D labelling in the rat spinal cord.

A comparison of the localization of the neurokinin 1 (NK1) receptor and nitric oxide synthase with calbindin D labelling in the lumbar spinal cord was carried out in the rat using immunocytochemistry. Considerable regional variations were observed. Application of the antibody to calbindin D resulted in dense staining in laminae I and II and light staining in the other laminae. Occasional scattered cells were seen in the deep laminae and in the lamina X, the ventral horn and the lateral spinal nucleus. The results indicate that neurones expressing calbindin D, NK1 receptor and NOS are three separate populations in the dorsal horn of the lumbar spinal cord.

Animals↗
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↗
PubMed1987

Chicken intestinal 28-kilodalton calbindin-D: complete amino acid sequence and structural considerations.

The complete primary structure of the cholecalciferol-induced chicken intestinal 28-kDa calbindin-D is reported. It is a single-chain polypeptide consisting of 261 amino acid residues (computed Mr = 30,042) and is blocked at the amino terminus. Tryptic digestion of the S-pyridylethylated protein followed by HPLC peptide mapping and automated sequence determination provided the bulk of the sequence information. Subsequent chymotryptic and Staphylococcus aureus V8 protease cleavages yielded the sequences of several additional regions as well as extensive overlapping of the tryptic peptides. The primary structure shows six homologous regions of sequence based on the EF-hand concept of calcium binding, four of which are predicted to actually bind calcium. Aside from these regions, there is no overall structural identity or apparent similarity with the mammalian calbindins (9 kDa), calmodulin, or troponin C. It is predicted that the secondary structure of 28-kDa calbindin-D is significantly different from the other proteins of this class, which bind four calcium atoms.

Amino Acid Sequence↗
PubMed2002

Human-specific organization of primary visual cortex: alternating compartments of dense Cat-301 and calbindin immunoreactivity in layer 4A.

There is evidence that the cortical anatomy of the magnocellular (M) visual pathway, which carries information about motion and luminance contrast, was modified in human evolution. Recent results indicate that layer 4A of humans contains a meshwork of tissue bands that stain densely for nonphosphorylated neurofilament (NPNF), a protein that is preferentially expressed in elements of the M pathway, whereas apes and monkeys lack a comparable pattern. Here we examined the distribution of staining for Cat-301 -- a monoclonal antibody well established to stain M-related structures preferentially -- in area V1 of humans, apes (chimpanzees, orangutan), Old World monkeys (macaques) and New World monkeys (spider monkeys, squirrel monkeys). Single-staining experiments, using a peroxidase-tetramethylbenzidine (TMB) reaction, revealed alternating zones of dark and light staining for Cat-301 in layer 4A of humans, similar to those observed with NPNF. Double-staining studies in humans revealed that Cat-301-immunoreactive somas and neuropil were localized within the same tissue bands that stained strongly for NPNF and, furthermore, that these bands alternated with irregularly shaped territories that stained very strongly for calbindin. Nonhuman primates, by contrast to humans, displayed weak Cat-301 and calbindin staining in layer 4A. The co-localization of Cat-301 and NPNF in human layer 4A, and the weak staining for these molecules in layer 4A of other primates, suggests that the cortical representation of the M channel was modified in recent human evolution. The calbindin-rich compartments in human layer 4A cannot be related to a particular geniculostriate pathway on neurochemical grounds; they may constitute an interneuronal population that increased in human evolution.

Animals↗
PubMed1988

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k.

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.

Amino Acids↗
PubMed1993

Calbindin D-28k and parvalbumin immunoreactivity in the frontal cortex in patients with frontal lobe dementia of non-Alzheimer type associated with amyotrophic lateral sclerosis.

The morphology and distribution of local-circuit neurons (interneurons) were examined, by calbindin D-28k and parvalbumin immunocytochemistry, in the frontal cortex (area 8) in two patients with frontal lobe dementia of non-Alzheimer type associated with classical amyotrophic lateral sclerosis (ALS), and in seven normal cases. The density of calbindin D-28k immunoreactive cells was dramatically reduced in ALS patients, but the density of parvalbumin-immunoreactive neurons was preserved. Decreased density of calbindin D-28k-immunoreactive neurons, which are mainly located in the upper cortical layers, may interfere with the normal processing of cortico-cortical connections, whereas integrity of parvalbumin-immunoreactive cells may be associated with the preservation of the major inhibitory intracortical circuits in patients with frontal lobe dementia.

Adult↗
PubMed1999

Calbindin immunoreactivity in the auricular lobe and interauricular granular band of the cerebellum in bullfrogs.

Calcium binding protein (CaBP) immunoreactivity in the cerebellum of bullfrogs was examined, concentrating on cells associated with the auricular lobe. While anti-calretinin and anti-parvalbumin also immunoreacted with the same cell populations, anti-calbindin exhibited the most robust and typical pattern of immunostaining. Calbindin immunoreactivity was observed in various populations of cells in the auricular lobe and interauricular granular band of the cerebellum, in the cerebellar peduncle, and in a bundle of interauricular commissural fibers which course through the dorsal, marginal, part of the molecular layer. Cells in the granular layer of the ventral part (i.e., corpus cerebelli) of the cerebellar plate were not CaBP-immunoreactive, nor were any fibers in the molecular layer of this cerebellar region. We believe that axons of CaBP-immunoreactive granule-like cells of the auricular lobes contribute to the formation of the interauricular fiber bundle, which corresponds to the lateral commissure of urodele amphibians. The pattern of calbindin immunoreactivity in the auricular lobes and marginal part of the cerebellar plate provides additional evidence that this cerebellar compartment, which is already present in tadpoles, has a distinct origin, biochemical characterization and connectivity and is separate from the compartment that forms the corpus cerebelli of frogs during metamorphosis.

Animals↗
PubMed1989

Calbindin-D immunolocalization in developing chick thyroid: a light and electron microscopic study.

Antiserum to calbindin-D, a 28 KD vitamin D-dependent calcium binding protein, was used to localize the protein immunocytochemically in developing chick thyroid by both light and electron microscopy. The protein first appeared in future follicular cells of developing thyroid tissue from 8-day-old embryos. The number of calbindin-D-containing cells increased rapidly to a near-plateau level at day 10; this concentration was sustained until day 15, and then declined to an undetectable level just before hatching. The protein was distributed throughout organelle-free areas of the follicular cell cytoplasm and extended into the nucleus; it was not present in the follicular colloid. Comparison of the time course of changes in calbindin-D content with known differentiative changes taking place in follicular cells suggests that the protein may function in some yet to be determined mechanism related to normal development of the thyroid.

Animals↗
PubMed1995

Calbindin-D9K gene expression in rat chorioallantoic placenta is not regulated by 1,25-dihydroxyvitamin D3.

The aim of this study was to investigate whether 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, regulates the expression of rat placental calbindin-D9K mRNA. One group of rats (-D) was fed a vitamin D-deficient diet before and during pregnancy, whereas a second group (+D) was fed a vitamin D-replete diet over the same period. Animals were killed on d 21 of gestation, and plasma concentrations of 1,25(OH)2D3 for +D and -D animals were significantly (p < 0.05) different (260 +/- 78 and 39 +/- 8 pM in maternal plasma and 122 +/- 39 and 42 +/- 10 pM in fetal plasma, respectively; mean +/- SE, n = 4-5). Vitamin D deficiency had no effect on placental weight, fetal weight, fetal ashed weight, fetal calcium accretion, or the maternofetal calcium gradient. Hybridization of RNA from maternal duodena (used as a positive control tissue) and placentas of +D and -D rats with a rat calbindin-D9K cDNA revealed a single 0.6-kb transcript in both tissues. The abundance of this transcript was markedly lower (p = 0.06) in the duodena of -D compared with +D rats (mean change -68 +/- 9%) but there was no difference between the placentas of the two groups (mean change +13 +/- 22%). These changes were significantly (p < 0.05) different between the two tissues and the response of each tissue to vitamin D deficiency was significantly different (p < 0.01). These data indicate that 1,25(OH)2D3 does not regulate the expression of calbindin-D9K mRNA in rat placenta.

Allantois↗
PubMed1993

Parvalbumin and calbindin D-28K immunoreactivity in central ganglioglioma and dysplastic gangliocytoma of the cerebellum. Report of two cases.

Calbindin D-28K and parvalbumin immunocytochemistry were used in the study of central ganglionic cell tumors. Most neurons in the ganglioglioma were immunoreactive to calbindin D-28K, but a few cells were labeled with antibodies against parvalbumin. In contrast, most cells in dysplastic gangliocytoma of the cerebellum were parvalbumin immunoreactive, but fewer reacted with anti-calbindin antibodies. These latter cells had two or three dendrites with claw-shaped terminals and axons with recurrent collateral branches and varicose terminals filled with strings and buttons. These observations suggest that central ganglionic cell tumors, including dysplastic gangliocytoma of the cerebellum, are composed of neurons which, on the basis of their calcium-binding protein content, have particular metabolic and electrophysiological properties.

Adolescent↗
PubMed1999

Calbindin and calmodulin localization in the developing vestibular organ of the musk shrew (Suncus murinus).

The distribution of the Ca(2+)-binding proteins, calbindin and calmodulin, in the adult inner ear has been described previously. We undertook immunohistochemical investigation of developmental changes in the distribution of calbindin and calmodulin in the vestibular organ of the musk shrew (Suncus murinus). Expression of calbindin was seen first in the hair cells and the vestibular ganglion on gestational day (GD) 19, in nerve fibres on GD23 and in the otoconia on GD26. On GD19 calmodulin was demonstrable only in the hair cells. On GD26 both Ca(2+)-binding proteins showed a distribution of immunoreactivity in hair cells similar to that seen in adults. The developmental differences in distribution of these binding proteins may suggest different roles in the vestibule. Additionally, Ca(2+)-binding proteins may be a useful index of hair cell maturity.

Animals↗
PubMed1999

Ionization behavior of acidic residues in calbindin D(9k).

The ionization state of seven glutamate residues, one aspartate, and the C-terminal alpha-COOH group in bovine apo calbindin D(9k) has been studied by measurement and modeling of the pH titration curves and apparent pK(a) values. The observed pK(a) ranged from 3.0 to 6.5. Most of the observed acidic groups were half-ionized at lower pH values than those in unstructured proteins. As a rule, the ionization equilibria extended over a wider pH range than in the case of unperturbed single titrations, indicating a complex influence of protein charges on the charge state of each individual residue. Glu17, which is a backbone Ca(2+)-ligand in the N-terminal binding loop of calbindin D(9k), was half-protonated at pH 3.6 but manifested biphasic titration with apparent pK(a) values of 3.2 and 6.5. Complementary Monte Carlo simulations of the titration process and pK(a) values of the acidic groups in calbindin D(9k) reproduce the experimentally observed titration features, except for the pronounced double titration of Glu17. Discrepancies between the results from direct measurement and from modeling may be partly caused by changes in the protein structure when the net charge changes from -8 to +11 over the isoelectric point at pH 5. Proteins 1999;37:106-115.

Animals↗
PubMed2000

pK(a) calculations of calbindin D(9k): effects of Ca(2+) binding, protein dielectric constant, and ionic strength.

Calbindin is a small (75 residues) helix-loop-helix ("EF-hand") calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca(2+) ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants K(a) (pK(a) = -log K(a)) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pK(a)'s. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pK(a) values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca(2+) ions increases the pK(a) values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca(2+) binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pK(a)'s. For the apo structure, passable values for the pK(a)'s of these ligating groups could be determined if the structure was allowed to relax upon ion removal.

Binding Sites↗
PubMed2002

Cholinergic innervation of parvalbumin- and calbindin-containing neurones in the hippocampus during postnatal development of the rat brain.

Immunohistochemical study of the cholinergic innervation of the parvalbumin- and calbindin-containing cells in the hippocampus was conducted on 30 rat brains of various postnatal ages: P0, P4, P7, P14, P21, P30, P60 and P180. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and parvalbumin (PV) or calbindin (CB) were analysed using confocal laser-scanning microscope. Obtained data demonstrate that the pattern of cholinergic innervation of calbindin- and parvalbumin-immunoreactive hippocampal neurones shows some differences. During development as well as in the adult species cholinergic terminals preferentially innervate CB-containing neurones, while cholinergic terminals on PV-containing cells were observed rarely. Cholinergic endings on the CB-ir neurones are localised both on their somata and dendrites, whereas on PV-ir cells they form synaptic contact predominantly with processes. In spite of the unquestionable cholinergic influence particularly on CB-ir cells, the number of cholinergic endings suggests that this input seems not to be crucial for the activity of the studied cell populations.

Animals↗
PubMed2003

Calbindin in cerebellar Purkinje cells is a critical determinant of the precision of motor coordination.

Long-term depression (LTD) of Purkinje cell-parallel fiber synaptic transmission is a critical determinant of normal cerebellar function. Impairment of LTD through, for example, disruption of the metabotropic glutamate receptor-IP3-calcium signaling cascade in mutant mice results in severe deficits of both synaptic transmission and cerebellar motor control. Here, we demonstrate that selective genetic deletion of the calcium-binding protein calbindin D-28k (calbindin) from cerebellar Purkinje cells results in distinctly different cellular and behavioral alterations. These mutants display marked permanent deficits of motor coordination and sensory processing. This occurs in the absence of alterations in a form of LTD implicated in the control of behavior. Analysis of synaptically evoked calcium transients in spines and dendrites of Purkinje cells demonstrated an alteration of time course and amplitude of fast calcium transients after parallel or climbing fiber stimulation. By contrast, the delayed metabotropic glutamate receptor-mediated calcium transients were normal. Our results reveal a unique role of Purkinje cell calbindin in a specific form of motor control and suggest that rapid calcium buffering may directly control behaviorally relevant neuronal signal integration.

Action Potentials↗