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PubMed1996

Transient neuronal population of the internal capsule in the developing human cerebrum.

The present study reveals the presence of a distinct group of cells, resembling reticular thalamic neurones, in the internal capsule during fetal development. This cell population rapidly decreases in size during early infancy and few cells are apparent in the 1-year-old infant. Internal capsule cells are well differentiated, multipolar or polymorphous, AChE (acetylcholinesterase)-reactive neurones. The following specific molecular markers were demonstrated in the neurones of the internal capsule: MAP2 (microtubule-associated protein 2), somatostatin, calbindin-D28K and p75 low-affinity NGF (nerve growth factor) receptor. A group of neurones described here corresponds to the perireticular thalamic nucleus found in certain mammalian species, hitherto unidentified in the primate brain, which may play an important role during development.

Acetylcholinesterase↗
PubMed1992

Differential Ca2+ binding properties in the human cerebellar cortex: distribution of parvalbumin and calbindin D-28k immunoreactivity.

The distribution of the Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CaBP) was investigated in the human cerebellar cortex. Purkinje cells contain both PV and CaBP. PV but not CaBP stains stellate and basket cells in the molecular layer. In the granular layer Golgi neurons can be subdivided into a majority, devoid of both Ca(2+)-binding proteins, and a scanty population which appears to be PV- and CaBP-immunoreactive. Thus GABAergic neurons in the human cerebellar cortex show selective differences in their Ca(2+)-binding properties, and these differences might reflect a heterogeneity in the processing of Ca(2+)-mediated events.

Aged↗
PubMed1995

The effect of insulin and insulin-like growth factor-1 on the expression of calretinin and calbindin D-28k in rat embryonic neurons in culture.

In a primary culture of rat embryonic neurons, insulin (5 mg/ml) promoted neurite formation between neuron clusters in serum/glial-free simplified medium with a high concentration of transferrin (100 micrograms/ml). Insulin growth factor-1 (IGF-1) exerted a similar effect in the same culture system at a lower dose (100 ng/ml) of insulin. Calretinin has recently been identified as a calcium binding protein expressed predominantly in sensory neurons, which has six calcium binding domains and shows molecular similarity to calbindin D-28k, an intestinal calcium transporter protein also found in the CNS. The effects of insulin and IGF-1 on the expression of calretinin and calbindin D-28k were investigated in rat embryonic neuronal cell culture. When cells were cultured for 2 days, insulin and IGF-1 promoted the expression of both proteins; when cultured for more than 2 days, IGF-1 still exerted a growth factor effect, but insulin decreased the expression of calretinin. Using the present culture system, we demonstrated that the effects of closely related molecules, insulin and IGF-1 which share receptors reciprocally, differed in calretinin and calbindin D-28k expression. Therefore, these two calcium binding proteins may play different physiological roles in the nervous system based either on different molecular mechanisms or switching roles at different stages of CNS development. Amplification based on Western blot employing the streptoavidin-horseradish peroxidase method was applied to detect calretinin and calbindin D-28k in the culture system by an immunoblotting technique.

Animals↗
PubMed2003

Calcium dynamics, buffering, and buffer saturation in the boutons of dentate granule-cell axons in the hilus.

The axons of dentate gyrus granule cells form synapses in the hilus. Ca(2+) signaling was investigated in the boutons of these axons using confocal fluorescence imaging. Boutons were loaded with various concentrations of the Ca(2+) indicator Oregon Green BAPTA-1 by patch-clamping the cell bodies and allowing the dye to diffuse into the axon. Resting free [Ca(2+)] started at 74 nm, rose to approximately 1 microm immediately after an action potential, and then decayed to rest with a time constant of 43 msec (all extrapolated to a dye concentration of zero). Action potential-induced [Ca(2+)] rises were smaller in larger boutons, consistent with a size-independent Ca(2+) channel density of 45/microm(2). Action potential-induced [Ca(2+)] changes varied with dye concentration in a manner consistent with kappa(E) approximately 20 for the ratio of endogenous buffer-bound Ca(2+) to free Ca(2+). During trains of action potentials, [Ca(2+)] increments summed supralinearly by more than that expected from dye saturation. The amount of endogenous Ca(2+) buffering declined as [Ca(2+)] rose, and this saturation indicated a buffer with a dissociation constant of approximately 500 nm and a concentration of approximately 130 microm. This is similar to the dissociation constant of calbindin-D28K, a Ca(2+)-binding protein that is abundant in dentate granule cells. Thus, calbindin-D28K is a good candidate for the Ca(2+) buffer revealed by these experiments. The saturation of endogenous buffer can generate short-term facilitation by amplifying [Ca(2+)] changes during repetitive activity. Buffer saturation may also be relevant to the presynaptic induction of long-term potentiation at synapses formed by dentate granule cells.

Action Potentials↗
PubMed1995

Calmodulin, calbindin-D28k, calretinin and neurocalcin in rat olfactory bulb during postnatal development.

Odorant stimulation of receptor cells results in a calcium influx that activates the transduction pathway. The olfactory neurons extend axons to the olfactory bulb where they synapse onto mitral cells. Ca(2+)-acceptors also may participate in subsequent processing of olfactory information. The present study describes the distribution of calmodulin, calretinin, calbindin-D28k and neurocalcin during rat main olfactory bulb development. From postnatal day 1 (P1) we observed in the olfactory nerve layer a thin external bundle containing calbindin and calretinin whereas calmodulin was present in a large internal bundle. In tufted cells, neurocalcin immunoreactivity was detected at P10 and increased until P20. In mitral cells calmodulin was intensively immunoreactive at P1 but decreased during development to disappear at adulthood whereas calretinin was weakly labelled at P1 but raised in intensity until P20. In granule cells calbindin-D28k and calretinin were detected from P1. Giant neurons were positive for both calretinin and calbindin-D28k from postnatal day 20.

Aging↗
PubMed2001

The KCl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in the rat hippocampus.

Immunocytochemical visualization of the neuron-specific K+/Cl- cotransporter, KCC2, at the cellular and subcellular level revealed an area- and layer-specific diffuse labelling, and a discrete staining outlining the somata and dendrites of some interneurons in all areas of the rat hippocampus. KCC2 was highly expressed in parvalbumin-containing interneurons, as well as in subsets of calbindin, calretinin and metabotropic glutamate receptor 1a-immunoreactive interneurons. During the first 2 postnatal weeks, an increase of KCC2 staining was observed in the molecular layer of the dentate gyrus, correlating temporally with the arrival of entorhinal cortical inputs. Subcellular localization demonstrated KCC2 in the plasma membranes. Immunoreactivity in principal cells was responsible for the diffuse staining found in the neuropil. In these cells, KCC2 was detected primarily in dendritic spine heads, at the origin of spines and, at a much lower level on the somata and dendritic shafts. KCC2 expression was considerably higher in the somata and dendrites of interneurons, most notably of parvalbumin-containing cells, as well as in the thorny excrescences of CA3 pyramidal cells and in the spines of spiny hilar and stratum lucidum interneurons. The data indicate that KCC2 is highly expressed in the vicinity of excitatory inputs in the hippocampus, perhaps in close association with extrasynaptic GABAA receptors. A high level of excitation is known to lead to a simultaneous net influx of Na+ and Cl-, as evidenced by dendritic swelling. KCC2 located in the same microenvironment may provide a Cl- extrusion mechanism to deal with both ion and water homeostasis in addition to its role in setting the driving force of Cl- currents involved in fast postsynaptic inhibition.

Animals↗
PubMed1998

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Aging↗
PubMed2006

Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38.

Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.

Animals↗
PubMed1997

Topographic localization of calretinin, calbindin, VIP, substance P, CCK and metabotropic glutamate receptor immunoreactive neurons in the supramammillary and related areas of the rat.

A detailed description of the localization of neurons containing various neuropeptides in the supramammillary complex (SUM) is provided. Further, the neurochemical character of supramammillohippocampal and supramammilloseptal projecting neurons was investigated. The following experiments were performed: (a) immunocytochemistry for each of the eight different neuropeptides investigated, in animals pretreated or not with colchicine, and perfused in fixative containing or lacking acrolein; (b) a thorough mapping study of the localization of immunolabelled neurons at three rostrocaudal levels; (c) double-tracing retrograde labelling for two-directional neuronal projections combined with immunocytochemistry, to study neurochemical character of the projecting neurons. The observations are: (1) each type of immunolabelled elements, such as calretinin, calbindin, VIP, substance P, CCK and metabotropic glutamate receptor 1a immunopositive neurons has a characteristic localization; (2) no parvalbumin- and enkephalin-containing neurons are present in the SUM; and (3) a small population of calretinin-containing and a small number of calretinin-negative supramammillohippocampal neurons located in the lateral area also project to the medial septum-diagonal band region of the septal complex.

Animals↗
PubMed1998

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia.

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D28, and s-100. Cultured cells or tissues were incubated with 10 microM BODIPY forskolin for 30 min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D28 (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.

Adenylyl Cyclases↗
PubMed2003

Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex.

Calbindin-D(28k) (CB), calretinin (CRT), and parvalbumin (PV) are high-affinity cytosolic calcium (Ca(2+)) binding proteins (CBP) that have been found to regulate intracellular calcium concentrations in neurons through their buffering capacity and to protect neurons from insults that induce elevations of intracellular Ca(2+). In earlier studies we observed a substantial and neurochemically specific loss of CB from the human basal forebrain cholinergic neurons (BFCN) in the course of normal aging. In the present experiments we expanded our investigation of age-related changes in calcium binding proteins in the human brain by investigating the status of CB-, CRT-, and PV-positive neurons in 17 cortical areas. There was a trend toward a decrease in the number of CB-immunoreactive neurons in all areas studied. However, this trend reached significance in only 4 areas in which the loss of CB-positive neurons ranged between 20 and 46%. Immunoreactivity for CRT was also decreased in many areas and this difference reached significance in three regions (26-37%). Cortical neurons displaying PV immunoreactivity did not show an age-related change. Comparison with other neurochemically specific cortical neurons indicated a similar age-related loss of nonphosphorylated neurofilament and NADPH-d activity in only a few cortical areas. In contrast, neuronal acetylcholinesterase activity was increased in a few cortical areas. These observations indicate that loss of CBP-positive neurons occurs in restricted cortical regions and is not a specific change as other neurochemically specific neurons also display restricted age-related changes. Furthermore, the age-related changes in cortical CBP-positive neurons appear to be considerably smaller than similar changes in the BFCN. The age-related depletion of CBPs is likely to deprive neurons from the capacity to buffer intracellular calcium and thus to leave them vulnerable to pathological processes that can cause increased intracellular calcium and lead to their degeneration.

Acetylcholinesterase↗
PubMed2002

Myo-inositol monophosphatase is an activated target of calbindin D28k.

Calbindin D(28k) (calbindin) is a member of the calmodulin superfamily of Ca(2+)-binding proteins. An intracellular target of calbindin was discovered using bacteriophage display. Human recombinant calbindin was immobilized on magnetic beads and used in affinity purification of phage-displayed peptides from a random 12-mer peptide library. One sequence, SYSSIAKYPSHS, was strongly selected both in the presence of Mg(2+) and in the presence of Ca(2+). Homology search against the protein sequence data base identified a closely similar sequence, ISSIKEKYPSHS, at residues 55-66 in myo-inositol-1(or 4)-monophosphatase (IMPase, EC ), which constitute a strongly conserved and exposed region in the three-dimensional structure. IMPase is a key enzyme in the regulation of the activity of the phosphatidylinositol-signaling pathway. It catalyzes the hydrolysis of myo-inositol-1(or 4)-monophosphate to form free myo-inositol, maintaining a supply that represents the precursor for inositol phospholipid second messenger signaling systems. Fluorescence spectroscopy showed that isolated calbindin and IMPase interact with an apparent equilibrium dissociation constant, K(D), of 0.9 microm. Both apo and Ca(2+)-bound calbindin was found to activate IMPase up to 250-fold, depending on the pH and substrate concentration. The activation is most pronounced at conditions that otherwise lead to a very low activity of IMPase, i.e. at reduced pH and at low substrate concentration.

Amino Acid Sequence↗
PubMed1996

Localization and regulation by vitamin D of calcium transport proteins in rabbit cortical collecting system.

The 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced expression of Na+/Ca2+ exchanger, Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase), and calbindin-D28k was investigated in the rabbit distal nephron. Immunocytochemical studies in rabbit kidney sections revealed colocalization of the three Ca2+ transport proteins in the majority of cells in the distal nephron, including connecting tubules and cortical collecting ducts. Subsequently, rabbit connecting and cortical collecting tubule cells were immunodissected and cultured on permeable supports. Immunocytochemical analysis of the cultured cells by confocal microscopy revealed that Na+/Ca2+ exchanger and Ca(2+)-ATPase were present at the basolateral membrane, whereas calbindin-D28k was evenly distributed throughout the cytosol. Concomitant with an increase in Ca2+ transport, 1,25(OH)2D3 increased calbindin-D28k protein and RNA content two- to threefold, as determined by Northern and Western blotting. By contrast, neither Na+/Ca2+ exchanger nor Ca(2+)-ATPase RNA or protein content was noticeably altered. Our findings suggest that 1,25(OH)2D3 stimulation of transcellular Ca2+ transport in primary cultures of rabbit cortical collecting system cells involves an increase in the gene expression of calbindin-D28k but not of Na+/Ca2+ exchanger and Ca(2+)-ATPase.

Animals↗
PubMed1992

Regulation of avian calbindin-D28K gene expression in primary chick kidney cells: importance of posttranscriptional mechanisms and calcium ion concentration.

Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.

Animals↗
PubMed2004

The distribution of P2X5 purinergic receptors in the enteric nervous system of mouse.

The distribution of the P2X(5) purinoceptor in the enteric nervous system of the mouse was studied by immunohistochemistry. P2X(5) receptor immunoreactivity was widely distributed in myenteric and submucosal plexuses throughout the gastrointestinal tract. In myenteric plexuses, immunoreactivity for the P2X(5) receptor was observed in nerve fibres that enveloped ganglion cell bodies, and possibly on glial cell processes. P2X(5) receptor immunoreactivity was colocalised with vasoactive intestinal peptide and surrounded ganglion cells that contained calretinin, calbindin or nitric oxide synthase. In the submucous plexus, P2X(5) receptor immunoreactivity occurred throughout the cytoplasm and on the surface membranes of the nerve cells. Double-labelling studies showed that 22%, 9%, 6% and 68% of P2X(5) receptor-immunoreactive neurones were also immunoreactive for calretinin, calbindin, nitric oxide synthase and vasoactive intestinal peptide, respectively. Thus, the P2X(5) receptor subunit is expressed in specific functional groups of neurones. P2X(2) and P2X(3) receptors were also present in the mouse enteric plexuses but no immunoreactivity for P2X(1), P2X(4) or P2X(6) receptors was found.

Animals↗
PubMed2005

Mono- and dual-frequency fast cerebellar oscillation in mice lacking parvalbumin and/or calbindin D-28k.

Calbindin is a fast Ca2+-binding protein expressed by Purkinje cells and involved in their firing regulation. Its deletion produced approximately 160-Hz oscillation sustained by synchronous, rhythmic Purkinje cells in the cerebellar cortex of mice. Parvalbumin is a slow-onset Ca2+-binding protein expressed in Purkinje cells and interneurons. In order to assess its function in Purkinje cell firing regulation, we studied the firing behavior of Purkinje cells in alert mice lacking parvalbumin (PV-/-), calbindin (CB-/-) or both (PV-/- CB-/-) and in wild-type controls. The absence of either protein resulted in Purkinje cell firing alterations (decreased complex spike duration and pause, increased simple spike firing rate) that were more pronounced in CB-/- than in PV-/- mice. Cumulative effects were found in complex spike alterations in PV-/- CB-/- mice. PV-/- and CB-/- mice manifested approximately 160-Hz oscillation that was sustained by Purkinje cells firing rhythmically and synchronously along the parallel fiber axis. This oscillation was dependent on GABA(A), N-methyl-D-aspartate and gap junction transmission. PV-/- CB-/- mice exhibited a dual-frequency (110 and 240 Hz) oscillation. The instantaneous spectral densities of both components were inversely correlated. Simple and complex spikes of Purkinje cells were phase-locked to one of the two oscillation frequencies. Mono- and dual-frequency oscillations presented similar pharmacological properties. These results demonstrate that the absence of the Ca2+ buffers parvalbumin and calbindin disrupts the regulation of Purkinje cell firing rate and rhythmicity in vivo and suggest that precise Ca2+ transient control is required to maintain the normal spontaneous arrhythmic and asynchronous firing pattern of the Purkinje cells.

2-Amino-5-phosphonovalerate↗
PubMed2001

Long-range effects on calcium binding and conformational change in the N-domain of calmodulin.

Proteins within the EF-hand protein family exhibit different conformational responses to Ca(2+) binding. Calmodulin and other members of the EF-hand protein family undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k (Clb), bind Ca(2+) without a significant change in conformation. Here, we investigate the effects of replacement of a leucine at position 39 of the N-terminal domain of calmodulin (N-Cam) with a phenylalanine derived from Clb. This variant is studied alone and in the context of other mutations that affect the conformational properties of N-Cam. Strikingly, the introduction of Phe39, which is distant from the calcium binding sites, leads to a significant enhancement of Ca(2+) binding affinity, even in the context of other mutations which trap the protein in the closed form. The results yield novel insights into the evolution of EF-hand proteins as calcium sensors versus calcium buffers.

Amino Acid Sequence↗
PubMed2003

The distribution of calcium buffering proteins in the turtle cochlea.

Hair cells of the inner ear contain high concentrations of calcium-binding proteins that limit calcium signals and prevent cross talk between different signaling pathways during auditory transduction. Using light microscope immunofluorescence and post-embedding immunogold labeling in the electron microscope, we characterized the distribution of three calcium-buffering proteins in the turtle cochlea. Both calbindin-D28k and parvalbumin-beta were confined to hair cells in which they showed a similar distribution, whereas calretinin was present mainly in hair-cell nuclei but also occurred in supporting cells and nerve fibers. The hair-cell concentration of calbindin-D28k but not of parvalbumin-beta increased from the low- to high-frequency end of the cochlea. Calibration against standards containing known amounts of calcium-buffering protein processed in the same fluid drop as the cochlear sections gave cytoplasmic concentrations of calbindin-D28k as 0.13-0.63 mm and parvalbumin-beta as approximately 0.25 mm, but calretinin was an order of magnitude less. Total amount of Ca 2+-binding sites on the proteins is at least 1.0 mm in low-frequency hair cells and 3.0 mm in high-frequency cells. Reverse transcription-PCR showed that mRNA for all three proteins was expressed in turtle hair cells. We suggest that calbindin-D28k and parvalbumin-beta may serve as endogenous mobile calcium buffers, but the predominantly nuclear location of calretinin argues for another role in calcium signaling. The results support conclusions from electrophysiological measurements that millimolar concentrations of endogenous calcium buffers are present in turtle hair cells. Parvalbumin-beta was also found in both inner and outer hair cells of the guinea pig cochlea.

Animals↗