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L Moccagatta

Publications and source records attributed to L Moccagatta.

4 recordsLinked to original sources
PubMed2002

Functional properties of EGFP-tagged skeletal muscle calcium-release channel (ryanodine receptor) expressed in COS-7 cells: sensitivity to caffeine and 4-chloro-m-cresol.

We constructed and expressed in COS-7 cells, three E-green fluorescent protein (EGFP) tagged recombinant skeletal muscle ryanodine receptors (RYR). EGFP was tagged to (i) the NH2-terminus (nEGFP-RYR(FL)) and to (ii) the COOH-terminus (cRYR(FL)-EGFP) of the full length RYR; we also tagged the EGFP to (iii) the NH2-terminus of a truncated version of the RYR (nEGFP-RYR(Bhat)) lacking the bulk of the protein. The fluorescent pattern EGFP with all three constructs colocalize with that of an endoplasmic reticulum (ER) membrane tracker fluorescent dye, indicating that the RYR constructs are targeted to ER membranes. Our results show that: (i) COOH-terminal tagging abolishes the sensitivity of the RYR to caffeine, whereas the presence of EGFP at the NH2-terminus does not affect caffeine sensitivity and (ii) 4-Cl-m-cresol sensitivity is lost both with the truncated nEGFP-RYR(Bhat) and the nEGFP-RYR(FL), while COOH-terminal tagging does not affect sensitivity to 4-chloro-m-cresol. The dose-response curves of caffeine-induced calcium release of nEGFP-RYR(FL) differ from those of the truncated nEGFP-RYR(Bhat). Maximal calcium release was approached at 10 mM caffeine with the nEGFP-RYR(FL), while cells expressing the nEGFP-RYR(Bhat) construct displayed a bell shaped curve and the maximal concentration for caffeine-induced calcium release was 5 mM. Equilibrium [3H]-ryanodine binding confirmed the calcium photometry data. Our results demonstrate that EGFP tagging modifies the pharmacological properties of RYR, and suggest that 4-chloro-m-cresol and caffeine act through different mechanisms and probably interact with different sites on the RYR calcium release channel.

Animals↗
PubMed2000

Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.

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 beta-hydroxylase, a member of the alpha-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 beta-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)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).

Alternative Splicing↗
PubMed1999

Pathways of cadmium influx in mammalian neurons.

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 microM Cd2(+). 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+/-5%) by 1 microM nimodipine and enhanced by 1 microM Bay K 8644. Concurrent application of nimodipine and omega-agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88+/-5%), whereas omega-conotoxin MVIIC (2 microM) reduced dR/dt by 24+/-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 omega-agatoxin IVA moderately reduced dR/dt (25+/-3%). NMDA-driven Cd2(+) entry was almost completely prevented by 1 mM Mg2+, 50 microM memantine, and 10 microM 5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with alpha-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.

Animals↗