1D Subunit Research Articles

The role of Ca 2+ channel modulation in the neuroprotective actions of estrogen in β-amyloid protein and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) cytotoxic models

Physiologically relevant concentrations of 17β-estradiol (E2) are neuroprotective in both β-amyloid protein 25–35 (Aβ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cytotoxicity in SK-N-SH cells. MPTP, but not Aβ, induces apoptosis in this cell line. The L-type calcium channel blocker nifedipine or decreased extracellular Ca 2+ concentration blocked Aβ-induced cell death, but not MPTP-induced cell death. Other blockers selective for different Ca 2+ channel subtypes had no effects on either Aβ or MPTP induced death. Western blot analysis for L-type Ca 2+ channel α 1-subunits demonstrated that Aβ increases the expression of the neuronal α 1C and α 1D subunits of L-type channels. Both E2 and nifedipine inhibit the increase in expression of these by Aβ. MPTP also increases expression of α 1C and α 1D, but the increases were not influenced by E2 or nifedipine. These observations suggested that Aβ cytotoxicity in SK-N-SH cells may involve increased availability of calcium to cells, whereas MPTP induced cytotoxicity does not require extracellular Ca 2+. Both cytotoxic models were associated with increased expression of Ca 2+ channel α 1 subunits, and neuroprotection associated with inhibition of that increase. These studies reveal that nifedipine, in addition to its direct action of nifedipine on Ca 2+ channels, may also protect neurons from Aβ toxicity through the suppression of the channel protein overexpression. A new action of dihydropyridines (DHPs) may be considered in the regulation of calcium homeostasis.

Giant paramagnetism in Li-doped Mo–S nanostructures

Magnetic measurements of Li-doped MoS 2− y nanostructures show a peculiarly large T-independent paramagnetic signal, which cannot be understood in terms of either Curie- or Pauli paramagnetism, or—for that matter—in terms of any simple spin-correlated state, such as antiferromagnetism or superparamagnetic spin clustering. This behaviour appears to be a result of a near-ideal one-dimensional (1D) strongly correlated state, due to the extraordinarily weak inherent coupling between 1D subunits (nanotubes or nanowires), which is an order of magnitude weaker even than in carbon nanotube ropes. In spite of clear evidence for strong electronic correlations from a giant paramagnetic susceptibility, no transition to an ordered state is observed down to very low temperatures and the system appears to be stabilised in a paramagnetic state by fluctuations characteristic of 1D systems. Down to 2 K there is no evidence of a low-temperature quantum critical point.

Regionally selective alterations in expression of the α 1D subunit (Ca v1.3) of L-type calcium channels in the hippocampus of aged rats

Calcium currents through the L-type voltage-sensitive calcium channel (L-VSCC) are increased in neurons of area CA1 of the hippocampus in aged rats and rabbits. Furthermore, increases in mRNA for the pore forming subunit α 1D (Ca v1.3) have been observed in the hippocampus of aged rats. We have studied the protein expression of the two pore forming subunits, α 1C (Ca v1.2) and α 1D, of L-VSCCs in the hippocampus of young and aged rats. Here we report selective age-related changes in expression of α 1D in the hippocampus. Specifically, we find that α 1D protein is increased in area CA1 of aged rats while it is decreased in area CA3. Our data suggest that the altered calcium currents seen in aged animals may be due, at least in part, to alterations in the expression of the α 1D subunit of the L-type calcium channel. These findings contribute to our understanding of the mechanisms responsible for changes in calcium homeostasis during aging.

Requirement for the L-type Ca(2+) channel alpha(1D) subunit in postnatal pancreatic beta cell generation.

Pancreatic beta cells are the source of insulin, which directly lowers blood glucose levels in the body. Our analyses of alpha(1D) gene-knockout (alpha(1D)(-/-)) mice show that the L-type calcium channel, alpha(1D), is required for proper beta cell generation in the postnatal pancreas. Knockout mice were characteristically slightly smaller than their littermates and exhibited hypoinsulinemia and glucose intolerance. However, isolated alpha(1D)(-/-) islets persisted in glucose sensing and insulin secretion, with compensatory overexpression of another L-type channel gene, alpha(1C). Histologically, newborn alpha(1D)(-/-) mice had an equivalent number of islets to wild-type mice. In contrast, adult alpha(1D)(-/-) mice showed a decrease in the number and size of islets, compared with littermate wild-type mice due to a decrease in beta cell generation. TUNEL staining showed that there was no increase in cell death in alpha(1D)(-/-) islets, and a 5-bromo-2' deoxyuridine-labeling (BrdU-labeling) assay illustrated significant reduction in the proliferation rate of beta cells in alpha(1D)(-/-) islets.

Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum

In the present study, we used immunohistochemistry and Western blot analysis to determine region-specific changes in the distribution of voltage-gated calcium channels (VGCCs) in aged rat cerebellum. Age-dependent changes in the staining intensity of the α 1C and α 1D subunits were prominent in the Purkinje cells, whereas there was no change in the expression of the α 1A and α 1B subunits. In the aged rat, in particular, immunoreactivity for the α 1C subunits were increased in the dendrites as well as in the cell bodies of Purkinje cells. On the other hand, decreases in immunoreactivity for α 1A and α 1D subunits were found in the molecular or granular layers. However, only α 1D subunit immunoreactivity was decreased in the aged cerebellum membrane by Western blot analysis that, while not addressing regional specificity, further confirmed an age-related decrease in α 1D subunit. These age-related changes in α 1D subunit expression might reflect a gradual loss of regulation for L-type Ca 2+ channels in the senescent period. The first demonstration of age-related alterations in VGCC expression may provide useful data for future investigations on aging and neurodegenerative diseases.

Fibroblast growth factor receptor 2 (FGFR2) in brain neurons and retinal pigment epithelial cells act via stimulation of neuroendocrine L-type channels (Cav1.3)

In contrast to the fibroblast growth factor receptor 1 (FGFR1), little is known about intracellular signaling of FGFR2. The signaling cascade of FGFR2 was studied using the perforated patch configuration of the patch-clamp technique in cultured rat retinal pigment epithelial (RPE) cells that express both FGFR1 and FGFR2. Interaction of signaling proteins was studied using immunoprecipitation techniques with membrane proteins from RPE cells and freshly isolated rat brain. When Ba(2+) currents through L-type channels were studied, extracellular application of bFGF (10 ng/ml) led to a shift of the steady-state activation to more negative values. In 50% of cells, an additional increase in maximal current amplitude was observed. This effect was blocked by the tyrosine kinase inhibitor lavendustin A (10(-5) M) but was not influenced by the FGFR1 blocker SU5402 (2 x 10(-5) M) or by the blocker for src-kinase herbimycin A (10(-5) M). Immunoprecipitation of FGFR2 led to coprecipitation of alpha 1D Ca(2+) channel subunits and precipitation of alpha 1D subunits led to coprecipitation of FGFR2. Immunoprecipitation of FGFR1 did not result in the coprecipitation with alpha 1D Ca(2+) channel subunits. The coprecipitation results were comparable when using brain tissue and RPE cells. The alpha 1D subunit-specific band were stained with antiphosphotyrosine antibodies. We conclude that FGFR2 acts via a different signaling cascade than FGFR1. This cascade involves an src-kinase-independent, close functional interaction of FGFR2 and the alpha subunit of neuroendocrine L-type channels.

Immunohistochemical study on the distribution of neuronal voltage-gated calcium channels in the rat cerebellum

Many neuronal processes are regulated by calcium influx through voltage-gated calcium channels (VGCCs), including protein phosphorylation, gene expression, neurotransmitter release, and firing patterns of action potential. In the present study, we have used anti-peptide antibodies directed against a unique sequence in rat α 1A, α 1B, α 1C and α 1D subunits of VGCCs to determine their cellular distribution in normal rat cerebellum. Throughout the molecular layer, immunoreactivity for α 1B and α 1D subunits were found in the cell bodies of basket and stellate cells as well as in the neuropil. In the Purkinje cells, only α 1C-IR was observed in the dendritic branches of Purkinje cells, whereas immunoreactivity for α 1B and α 1D subunits were rarely found in the cell bodies of Purkinje cells. Immunoreactivity for the α 1A, α 1B, and α 1D subunits were strong in the granule cell bodies, whereas α 1C-IR was not prominent in the cell bodies. In the cerebellar nuclei, a distinct band of punctate immunoreactivity for the α 1A, α 1B, α 1C, and α 1D subunits were observed. The overall results of the above localization study showed clearly that the α 1A, α 1B, α 1C and α 1D pore forming subunits of VGCCs have differential distribution in the rat cerebellum. The present studies may provide useful data for such future investigations to understand the role of calcium channels in neurological pathways.

Genomic structure of the regulatory region of the voltage-gated calcium channel alpha 1D.

In excitable and endocrine organs, calcium influxes through the L-type voltage-gated calcium channel (VGCC) which is composed of four (alpha 1, alpha 2, beta, and gamma) subunits. Temporal and spatial expression of calcium channel activity is regulated by the transcription of alpha 1 subunit. To elucidate the genomic organization of the VGCC alpha 1D subunit gene, a genomic clone was isolated from the human genomic library and its sequence was analyzed. A 12 kb genomic clone contained the 5'-flanking regulatory region and first two exons was selected and the initiation site for alpha 1D mRNA synthesis was examined by primer extension analysis. The major initiation site was found at the -523 NT position in the translation initiation site. The TATA box could not be found above the transcription initiation site. The CAT vector construct containing the 2.5 kb upstream region had high CAT activity on transfection to NG108-15 and PC12 cells, which confers the neuronal expression of the alpha 1D gene.

Up-regulation of α 1D Ca 2+ channel subunit mRNA expression in the hippocampus of aged F344 rats

There is growing evidence that alterations in calcium (Ca 2+) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca 2+ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca 2+ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca 2+ channel subunits. A significant aging-related increase in mRNA content for the α 1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the α 1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the β 1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in α 1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca 2+ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca 2+ homeostasis.

Increased expression of voltage-activated calcium channels in cultured hippocampal neurons from mouse trisomy 16, a model for Down syndrome

Calcium is an important second messenger that affects metabolic and physiological activities of developing and mature neurons. It has been reported that electrical activity is abnormal in cultured hippocampal and DRG neurons from the trisomy 16 (Ts16) mouse, a model for Down syndrome (trisomy 21—Ts21 in human). Whole-cell voltage-clamp, radiolabeled ligand binding techniques and mRNA measurements were used to study the effect of Ts16 on voltage-dependent calcium currents in cultured fetal hippocampal neurons from the Ts16 mouse. In neither Ts16 nor control diploid neurons were low-voltage-activated calcium currents detected. However, a high-voltage-activated (HVA) calcium current was identified and shown to be dihydropyridine sensitive. The density of this HVA calcium current was 80% greater in Ts16 neurons than in control. This difference correlated with a 70% increase in binding of radiolabeled dihydropyridine, PN200-110, a marker of L-type calcium channels. However, mRNA levels encoding the α 1C and α 1D subunits were unchanged in the Ts16 neurons. In contrast, mRNA level of the myo-inositol transporter, the gene for which is located on mouse chromosome 16, was elevated in Ts16 neurons due to a gene-dosage effect. Therefore, it is likely that posttranscriptional regulation of dihydropyridine-sensitive voltage-dependent calcium channels is abnormal in Ts16. As dihydropyridine sensitive HVA Ca channels are implicated in heterosynaptic long-term depression and long-term potentiation, the differences reported here, if also present in the Down syndrome brain, may contribute to mental retardation in that disorder.

Voltage-dependent binding and calcium channel current inhibition by an anti-alpha 1D subunit antibody in rat dorsal root ganglion neurones and guinea-pig myocytes.

1. The presence of calcium channel alpha 1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea-pig cardiac myocytes was demonstrated using the reverse transcriptase-polymerase chain reaction. 2. An antipeptide antibody targeted at a region of the voltage-dependent calcium channel alpha 1D subunit C-terminal to the pore-forming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3. Incubation of cells under depolarizing conditions for 2-4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 8644 (1 microM) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibody that had been preabsorbed with its immunizing peptide (100 micrograms ml-1). This was significantly (P < 0.05) larger than the enhancement by S-(-)-Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. These results demonstrate the presence of calcium channel alpha 1D subunits in rat DRG neurones and guinea-pig cardiac myocytes. They also show that amino acids 1417-1434 of the alpha 1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S-(-)-Bay K 8644 to enhance this current, indicating that it is an L-type current that is attenuated.

Transcriptional regulation of the neuronal L-type calcium channel alpha 1D subunit gene.

1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence.

Structure and functional expression of α1, α2, and β subunits of a novel human neuronal calcium channel subtype

The primary structures of human neuronal α 1, α 2, and β subunits of a voltage-dependent Ca 2+ channel were deduced by characterizing cDNAs. The α 1 subunit ( α 1 D ) directs the recombinant expression of a dihydropyridine-sensitive L-type Ca 2+ channel when coexpressed with the β ( β 2) and the α 2 ( α 2 b ) subunits in Xenopus oocytes. The recombinant channel is also reversibly blocked by 10–15 μM ω-conotoxin. Expression of the α 1D subunit alone, or coexpression with the α 2b subunit, did not elicit functional Ca 2+ channel activity. Thus, the β 2 subunit appears to serve an obligatory function, whereas the α 2b subunit appears to play an accessory role that potentiates expression of the channel. The primary transcripts encoding the α 1D, α 2, and β subunits are differentially processed. At least two forms of neuronal α 1D were identified. Different forms of α 2 and β transcripts were also identified in CNS, skeletal muscle, and aorta tissues.

ChemInform Abstract: THE ELECTRONIC STRUCTURE AND THE NATURE OF CHEMICAL BOND OF CRYSTALLINE LITHIUM SILICIDE (LI12SI7). A SEMIEMPIRICAL CRYSTAL ORBITAL (CO) APPROACH BASED ON THE SELF‐CONSISTENT‐FIELD (SCF) APPROXIMATION IN THE HARTREE‐FOCK (HF) SCHEME APPLIED TO ONE‐DIMENSIONAL (1D) SUBUNITS OF THE LI12SI7 SOLID

Chemischer InformationsdienstVolume 16, Issue 34 Physical Inorganic Chemistry ChemInform Abstract: THE ELECTRONIC STRUCTURE AND THE NATURE OF CHEMICAL BOND OF CRYSTALLINE LITHIUM SILICIDE (LI12SI7). A SEMIEMPIRICAL CRYSTAL ORBITAL (CO) APPROACH BASED ON THE SELF-CONSISTENT-FIELD (SCF) APPROXIMATION IN THE HARTREE-FOCK (HF) SCHEME APPLIED TO ONE-DIMENSIONAL (1D) SUBUNITS OF THE LI12SI7 SOLID M. C. + BOEHM, M. C. + BOEHMSearch for more papers by this authorR. RAMIREZ, R. RAMIREZSearch for more papers by this authorR. NESPER, R. NESPERSearch for more papers by this authorH. G. VON SCHNERING, H. G. VON SCHNERINGSearch for more papers by this author M. C. + BOEHM, M. C. + BOEHMSearch for more papers by this authorR. RAMIREZ, R. RAMIREZSearch for more papers by this authorR. NESPER, R. NESPERSearch for more papers by this authorH. G. VON SCHNERING, H. G. VON SCHNERINGSearch for more papers by this author First published: August 27, 1985 https://doi.org/10.1002/chin.198534004AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume16, Issue34August 27, 1985 RelatedInformation

The purification and N-terminal amino acid sequence analysis of the high molecular weight gluten polypeptides of wheat

Nine high molecular weight gluten subunits have been purified from cultivars of bread wheat ( Triticum aestivum). They have broadly similar amino acid compositions with 34–39 mol% Glx, 12–17 mol% Pro and 14–19 mol% Gly. Some differences in the contents of other amino acids were apparent; notably cysteine varied from 0.4 to 1.5 mol%. On the basis of these compositions, the peptide maps with V8 proteinase and N-terminal amino acid sequences the subunits could be divided into four groups. Two of these contained subunits encoded by structural loci on chromosomes 1A and 1B, respectively, while the other two probably represented the products of two subfamilies of genes at a single locus on chromosome 1D. The N-terminal amino acid sequences were homologous, but differed in substitutions of single amino acids and in deletions of three and seven amino acids. Two subunits purified from Triticum monococcum and Aegilops squarrosa had N-terminal sequences closely related or identical to those of the 1A subunits and one group of 1D subunits respectively. Two cysteine residues were present in the N-terminal sequences of all the subunits, but the distances between these were affected by the deletions and varied from 7 to 14 residues. The results are discussed in relation to the ability of the subunits to form elastic disulphide-linked aggregates, and the importance of these in the structure and functionality of gluten.