Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH 2 -terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl ␤-hydroxylase, a member of the ␣-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl ␤-hydroxylase result from alternative splicing of the same gene.The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium 45

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca2+ from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP3) receptors (InsP3Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP3R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion–induced calcium entry, whereas its NH2 terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.

Abstract:The influx of the toxic cation Cd2+ was studied in fura 2-loaded rat cerebellar granule neurons. In cells depolarized with Ca2+-free, high-KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 pM Cd"'. This increase was fully reversed by the Cd2+ chelator tetrakis(2-pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67 t 5%) by 1 pM nimodipine and enhanced by 1 pM Bay K 8644. Concurrent application of nimodipine and o-agatoxin IVA (200 nM) blocked Cd" permeation almost completely (88 ? 5%), whereas w-conotoxin MVllC (2 pM) reduced dRldt by 24 t 8%. These results indicate a primary role of voltage-dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and o-agatoxin IVA moderately reduced dR/dt (25 2 3%). NMDA-driven Cd"+ entry was almost completely prevented by 1 mM Mg2+, 50 pM memantine, and 10 pM5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with cr-amino-3-hydroxy-5-methylisoxazole-4-propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx. Key Words: Cadmium uptake-Calcium channel-Glutamate receptors-Neurotoxicity-Fura 2. J. Neurochem. 72, 21 54-21 61 (1 999).Cadmium is an extremely harmful transition metal ion whose presence as an environmental contaminant increased significantly with the development of modern industries. A very long biological half-life, -30 years in humans, contributes to its accumulation into living cells, causing various adverse effects. Renal damage follows chronic exposure to low doses of Cd2+ (Endo and Shaikh, 1993;Shaikh et al., 1995), and acute poisoning of lungs, ovaries, bones, and several other organs has been reported in the presence of high Cd2+ concentrations. Severe pathologies of the CNS are elicited by the high blood-brain barrier permeability to Cd2+ (Wong and Klaassen, 1982;Babitch, 1988;Sutoo, 1994;Choudhuri et al., 1996), with large lesions located primarily in white matter (Fern et al., 1996). Finally, carcinogenic effects may be induced by Cd2+ (De et al., 1990;Jin and Ringertz, 1990;Selypes et al., 1992), Moccagatta, and Carla MarchettiNazionale delle Ricerche, Genova, Italy through transcription of the protooncogenes c-jun and c-myc.The cellular mechanisms underlying cadmium toxicity are complex and still debated: Cd2+ affects intracellular Ca2+ binding proteins, including tubulin, troponin C, and calmodulin, binds human serum transferrin, disrupts mitochondrial respiration, and heavily interferes with the Ca2+ influxlefflux machinery.Cadmium is a potent inhibitor of voltage-dependent calcium channels (VDCCs) and blocks the high-voltage-activated, dihydropyridine (DHP)-sensitive calcium channel type (L-type) with an IC,, close to l p M . Cd2+ at 100 p L M f...

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