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The selective innervation by serotoninergic axons of calbindin-containing interneurons in the neocortex and hippocampus of the marmoset.

The serotoninergic input to the mammalian cerebral cortex originates in the median and the dorsal raphe nuclei. Median raphe neurons have been previously shown to give rise to beaded varicose axons which form dense pericellular arrays (baskets) surrounding the soma and the proximal dendrites of certain cortical neurons. In the present study, we have searched for specific markers characterizing the neurons of the marmoset neocortex and hippocampus surrounded by these thick varicose serotonin-containing fibers. The non-pyramidal nature of these neurons, suggested by their dendritic arborization, was correlated, in immunocytochemical experiments with double-labelling to demonstrate their surrounding serotonin-containing basket and their content of glutamic acid decarboxylase (GAD) or of the calcium-binding protein calbindin. Another calcium-binding protein common in numerous non-pyramidal cortical neurons, parvalbumin, was never found in neurons surrounded by serotonin-containing baskets. This organization was found in all areas of the neocortex and of the hippocampus where serotonin-containing baskets were present. One of the serotoninergic cortical inputs which originates from the brainstem tegmentum, traditionally described as "diffuse," proves to be highly selective in that a subset of its axons terminates preferentially on a subpopulation of inhibitory interneurons of the cerebral cortex. It may be emphasized that this subset of cortical interneurons has now been shown to be characterized not only by its axonal and dendritic arborization and its neurotransmitter, but also by a specific type of input which can modulate cortical function in a unique manner.

Animals↗

Complementary expression of parvalbumin and calbindin D-28k delineates subdivisions of the rabbit medial geniculate body.

The complementary pattern of immunohistochemical staining for the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) was used to delineate four major subdivisions of the rabbit medial geniculate body (MGB). PV immunoreactivity predominates in the ventral and medial divisions, whereas CB-immunoreactive cells characterize the dorsal and internal divisions. The ventral nucleus is strongly PV+ due to dense neuropil labeling and moderately labeled somata. The medial nucleus contains both medium-sized and large PV+ somata, as well as thick PV+ axons and terminals. The wedge-shaped internal nucleus composed of densely labeled CB+ cells, separates the dorsal and ventral nuclei rostrally, and expands caudally to encapsulate the posterior MGV. Large multipolar CB+ neurons with radiate dendrites characterize the dorsal nucleus. The differential expression of PV and CB also distinguishes the deep dorsal and superficial dorsal subnuclei in the dorsal division and a ventrolateral component in the ventral division. A comparison with studies of MGB connectivity in a variety of species suggests that PV immunoreactivity is highest in subdivisions that receive a substantial input from the central nucleus of the inferior colliculus and that project to primary auditory cortex. In contrast, CB immunoreactivity characterizes nuclei that receive input primarily from other sources, such as the paracentral nuclei of the inferior colliculus, the lateral tegmentum, and the spinal cord, and that project to secondary auditory areas. The ability of calcium-binding protein immunohistochemistry to delineate neuronal compartments across indistinct cytoarchitectonic borders makes it a powerful tool for guiding future connectional and physiological studies of the MGB.

Animals↗

Overexpression of calbindin D(28k) in dentate gyrus granule cells alters mossy fiber presynaptic function and impairs hippocampal-dependent memory.

Calcium is a key signaling ion for induction of synaptic plasticity processes that are believed to influence cognition. Mechanisms regulating activity-induced increases in neuronal calcium and related synaptic modifications are not fully understood. Moreover, involvement of specific synapses in discrete aspects of spatial learning remains to be elucidated. We used herpes simplex amplicons to overexpress calbindin D(28k) (CaBP) selectively in dentate gyrus (DG) granule cells. We then examined the effects on hippocampal network activity by recording evoked synaptic responses in vivo and in vitro and analyzing hippocampal-dependent behavior. Relative to Lac-Z- and sham-infected controls, CaBP overexpression increased mossy fiber (MF-CA3) excitatory postsynaptic potentials and reduced paired-pulse facilitation (PPF), suggesting an increase in presynaptic strength. Additionally, CaBP overexpression reduced long-term potentiation (LTP), caused a frequency-dependent inhibition of post-tetanic potentiation (PTP), and impaired spatial navigation. Thus, increasing CaBP levels selectively in the DG disrupts MF-CA3 presynaptic function and impairs spatial cognition. The results demonstrate the power of gene delivery in the study of the neural substrates of learning and memory and suggest that mossy fiber synaptic plasticity is critical for long-term spatial memory.

Animals↗

Effect of glucocorticoid administration on intestinal, renal, and cerebellar calbindin-D28K in chicks.

A radioimmunoassay for chick intestinal calcium-binding protein (calbindin-D28K, CaBP-28K) has been developed in our laboratory with a detection limit of 0.3 ng/ml. The values for CaBP-28K in vitamin D-deficient (-D) chicks ranged from a high value for the cerebellum (21,400 +/- 580 ng/mg protein) to a scarcely detectable level in the liver (19.6 +/- 2.2 ng/mg protein). After administration of vitamin D (vitamin D3 500 IU p.o. for 7 days) (+D), the levels of CaBP-28K increased in the duodenum (52,300 +/- 5,100 ng/mg protein), ileum (45,200 +/- 740 ng/mg protein), cerebellum (22,000 +/- 470 ng/mg protein), colon (15,200 +/- 330 ng/mg protein), and kidney (13,460 +/- 540 ng/mg protein). However, the increment in the level of CaBP-28K in each tissue after vitamin D administration was different; levels of CaBP-28K in the duodenum, ileum, and colon increased dramatically more than 200 times after vitamin D administration, whereas that in the kidney showed only a 2.5-fold increase and was unaltered in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mRNAs for avian intestinal calbindin-D28k and the 1,25-dihydroxyvitamin D3 receptor.

We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.

Animals↗

1,25(OH)2D3-dependent regulation of calbindin-D28k mRNA requires ongoing protein synthesis in chick duodenal organ culture.

Organ culture of 19-day-old chick embryo duodena was utilized to evaluate the mechanism of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent calbindin-D28k (CaBP) expression. Duodenal CaBP and 1,25(OH)2D3 receptor (VDR) expression were assessed by Western blot analysis, while CaBP and VDR mRNA levels were determined by Northern blot analysis. In untreated duodena, both VDR protein and mRNA were present, while CaBP protein and mRNA were undetectable. Treatment of cultured duodena with 25 nM 1,25(OH)2D3 resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [3H-1 beta]1 alpha,25(OH)2D3 in duodenal chromatin is rapid (< or = 30 min). Thus, the delayed accumulation of detectable CaBP mRNA cannot be explained by slow nuclear binding of 1,25(OH)2D3. The inclusion of 1.6 microM actinomycin D in the organ culture partially inhibited the 1,25(OH)2D3-regulated increase in CaBP mRNA, which implies that there is a transcriptional component involved in the increased CaBP mRNA levels. Similarly, quantitative polymerase chain reaction studies allowed the detection of CaBP pre-mRNA and mRNA sequences 1 h after hormone treatment, suggesting that CaBP gene transcription is initiated rapidly. Treatment of cultures with 36 microM cycloheximide 1 h prior to 1,25(OH)2D3 addition resulted in superinduction of VDR mRNA levels but sharply reduced CaBP steady-state mRNA levels. This dramatic reduction in CaBP mRNA reveals that 1,25(OH)2D3-mediated CaBP expression is dependent on ongoing protein synthesis. Thus, we propose that a labile auxiliary protein or other cofactor, which may or may not be 1,25(OH)2D3-dependent, is necessary for 1,25(OH)2D3-mediated CaBP gene transcription in chick duodena.

Animals↗

Immunohistochemical and hodological characterization of calbindin-D28k-containing neurons in the spinal cord of the turtle, Pseudemys scripta elegans.

Neurons and fibers containing the calcium-binding protein calbindin-D28k (CB) were studied by immunohistochemical techniques in the spinal cord of adult and juvenile turtles, Pseudemys scripta elegans. Abundant cell bodies and fibers immunoreactive for CB were widely and distinctly distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. In the dorsal horn, most CB-containing cells were located in close relation to the synaptic fields formed by primary afferents, which were not labeled for CB. Double immunohistofluorescence demonstrated distinct cell populations in the dorsal horn labeled only for CB or nitric oxide synthase, whereas in the dorsal part of the ventral horn colocalization of nitric oxide synthase was found in about 6% of the CB-immunoreactive cells in this region. Choline acetyltransferase immunohistochemistry revealed that only about 2% of the neurons in the dorsal part of the ventral horn colocalized CB, whereas motoneurons were not CB-immunoreactive. The involvement of CB-containing neurons in ascending spinal projections to the thalamus, tegmentum, and reticular formation was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Similar experiments demonstrated supraspinal projections from CB-containing cells mainly located in the reticular formation but also in the thalamus and the vestibular nucleus. The revealed organization of the neurons and fibers containing CB in the spinal cord of the turtle shares distribution and developmental features, colocalization with other neuronal markers, and connectivity with other tetrapods and, in particular with mammals.

Animals↗

Tissue-specific expression of calbindin-D28K gene during ontogeny of the chicken.

The vitamin D3-dependent calcium binding protein, calbindin-D28K (CaBP-D28K), plays an important role in transepithelial calcium translocation. To evaluate its role in chick embryonic calcium metabolism, steady-state levels of CaBP-D28K mRNA in various tissues of the chick embryo were determined by Northern blot and slot blot analyses, and CaBP-D28K concentrations in the examined tissues and plasma were estimated by RIA. High levels of CaBP-D28K mRNA were found in the embryonic kidney (mesonephros) on embryonic day (E) 10 and E12 and thereafter gradually decreased until hatching. CaBP-D28K mRNA levels were low in the yolk sac until E16 but increased on E18 and reached a maximum on E20. A steady increase in CaBP-D28K mRNA levels was observed in the cerebellum during the development from E10 to post-hatching. CaBP-D28K mRNA levels in the intestine were very low during the incubation period but significantly increased on days 1 and 7 after hatching. By Northern blot analysis, CaBP-D28K mRNA was barely detectable in liver, heart, and chorioallantoic membrane of the embryonic chick. Changes in immunoreactive CaBP-D28K of each tissue paralleled observed changes in mRNA levels. In plasma, measurable levels of CaBP-D28K were found as early as E8 and were stable until E18, when 6.5-fold increase was observed compared to E16. The highest level of CaBP-D28K in plasma was found on E20 and decreased after hatching. These temporal profiles of CaBP-D28K suggest that it may play an important role in the regulation of chick embryonic calcium homeostasis.

Aging↗

Bone morphogenetic protein-6 is a neurotrophic factor for calbindin-positive striatal neurons.

Bone morphogenetic proteins (BMPs) are a set of members of the transforming growth factor-beta superfamily recently described as promoting the differentiation of several neuronal populations within the basal ganglia. This study examined whether a member of this family, BMP-6, could exert neurotrophic effects on the neurons of the striatum, in which BMP-6 mRNA had been previously detected during development. Here we show that BMP-6 increases the number and differentiation of calbindin-positive neurons in vitro. Indeed, BMP-6 increased the total area, the perimeter, and the degree of arborization of this neuronal population. This trophic factor promoted dendritic growth without modifying axonal length or soma area. Furthermore, BMP-6 increased the number of glial fibrillary acidic protein-positive cells while decreasing the number of nestin-positive cells. The suppression of cell proliferation or glial development by the antimitotic fluorodeoxyuridine removed the effects on striatal neurons, suggesting the involvement of astroglial cells in the differentiation induced by BMP-6. The current results confirm the relevance of BMPs in the development of the striatum and emphasize the crucial importance of the trophic interaction between glial and neuronal cells.

Animals↗

Immunohistochemistry with anti-calbindin and anti-neurofilament antibodies in the cerebellum of methylazoxymethanol-treated mice.

Mice pups were injected with methylazoxymethanol at birth (MAM0) or on the fifth postnatal day (MAM5) and their cerebella were examined when adult. Immunohistochemistry with an antiserum directed against calbindin, a protein specific for Purkinje cells, was used to survey more easily Purkinje cell position and orientation. For a general view of basket cell axon distribution, we used a monoclonal antibody that recognized the phosphorylated form of the 200 kD constituent protein of neurofilaments, which is axon specific. The present results confirm that in MAM5 the cytoarchitecture was preserved, some Purkinje cells degenerated, and the pericellular basket around the Purkinje cells was apparently normal. In MAM0 animals, the Purkinje cells appeared malpositioned and disoriented, the pinceau around the Purkinje cell hillock was absent, but basket cell axons were present. This indicated that the absence of pinceau was not due to the absence of basket cells, but probably to alterations of cell interactions, which hindered the proper pericellular basket formation.

Animals↗

The baboon expresses the calbindin-D9k gene in intestine but not in uterus and placenta: implication for conservation of the gene in primates.

Expression of the Calbindin-D9k (CaBP-9k) gene was studied in the baboon. Northern blot analysis using a human CaBP-9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP-9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP-9k gene using human-derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP-9k intron A, including the presence a complete Alu-repetitive element. Most significantly, a 13 nucleotide long element at the 5' end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP-9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP-9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian-specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP-9k in these reproductive tissues.

Animals↗

Dominant expression of porcine Calbindin-D9k in the uterus during a luteal phase.

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue.

Animals↗

A model of pulmonary adenocarcinoma in transgenic mice expressing the simian virus 40 T antigen driven by the rat Calbindin-D9K (CaBP9K) promoter.

Lung cancer is the most frequent cause of cancer deaths. Its origin and development remain poorly understood, partly because of the lack of pertinent animal models. This study produced transgenic mice expressing the simian virus (SV) 40 T antigen (Tag) driven by a 1011 base-pair DNA fragment of the rat Calbindin-D9K (CaBP9K) promoter. All transgenic animals developed multifocal pulmonary tumours with pathological and ultrastructural features consistent with adenocarcinomas. Using immunohistochemistry, northern blot or western blot, tumours were found to express the transcription factor TTF-1, as well as specific markers of the peripheral airway Clara cells (CC10) and alveolar type II cells (surfactant proteins A, B, C, and D). This model, with its similarities to human adenocarcinoma, should be useful not only for addressing the mechanisms underlying the development and progression of lung cancer, but also for testing new therapeutic approaches.

Adenocarcinoma↗

Characterization of the N-terminal half-saturated state of calbindin D9k: NMR studies of the N56A mutant.

Calbindin D9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (1) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038-1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium-loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.

Animals↗

Structural basis for the negative allostery between Ca(2+)- and Mg(2+)-binding in the intracellular Ca(2+)-receptor calbindin D9k.

The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D9k were determined to 1.6 A and 1.9 A resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1H-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.

Allosteric Regulation↗

Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands.

Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.

Amino Acid Sequence↗

Focusing of the electrostatic potential at EF-hands of calbindin D(9k): titration of acidic residues.

Biological functions for a large class of calmodulin-related proteins, such as target protein activation and Ca(2+) buffering, are based on fine-tuned binding and release of Ca(2+) ions by pairs of coupled EF-hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK(a) values for all aspartic and glutamic acid residues in apo calbindin D(9k), a representative of calmodulin-related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca(2+) ligands with high pK(a) under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca(2+) ligand of each site, and that the potential is significantly more negative at the N-terminal binding site.

Animals↗

Design and synthesis of the pseudo-EF hand in calbindin D9K: effect of amino acid substitutions in the alpha-helical regions.

A series of 37-residue analogues of the pseudo-EF hand in bovine calbindin D9K has been synthesized by the solid phase method. In the presence of calcium an alpha-helical induction of up to 44% was observed for the peptide with the native sequence with a Kd for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two alpha-helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu-...Lys+ salt bridge or by changing the N-terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices. The mutants with the substitution of (Leu-30, Leu-32) to (Gln-30, Gln-32), (Gln-30, Ala-32), and (Ala-30,Ala-32) designed based on the strategy 1 do not show any affinity for calcium and have low alpha-helicity. The Leu-30 to Lys-30 mutant designed to form a salt bridge between the side chains of Glu-26 and Lys-30 has an apparent Kd for calcium of 6.8 mM. Kd of the N-terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the alpha-helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr-13, Leu-31 to Cys-13, Cys-31 and Tyr-13, Leu-31 to Cys-13, hCys-31, show apparent Kd values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest alpha-helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix-loop-helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.

Amino Acid Sequence↗