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Biomedical subjects

E Carafoli

Publications and source records attributed to E Carafoli.

At least 55 records · Page 3Linked to original sources
PubMed1997

Subcellular and tissue distribution, partial purification, and sequencing of calmodulin-stimulated Ca2+-transporting ATPases from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum).

The subcellular distribution of plasma-membrane-type Ca2+-transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner-membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)-enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+-transporting ATPase (molecular mass approximately 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116-kDa Ca2+-transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue-specific manner: the PM pump was dominant in leaves, while the inner-membrane pump was expressed at a higher level in the roots. For the purification of calmodulin-binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116 kDa was partially sequenced.

Adenosine Triphosphate↗
PubMed1997

Immunolocalization of the plasma membrane Ca2+ pump isoforms in the rat brain.

Ca2+ homeostasis in nerve cells is dependent on at least three mechanisms: Ca2+ channels, calcium-binding proteins and Ca2+ exchangers/pumps. Only limited information is available on the regional/cellular distribution of these Ca2+-regulating systems in the brain. The distribution of three of the isoforms of one of the systems, plasma membrane Ca2+-ATPase (PMCA), was analyzed in this study. Using antibodies against epitopes specific for each isoform, a map of the distribution of the pump in the whole brain was produced. The pump was mainly expressed in neurons and was apparently absent from glia cells. Isoform 1 was ubiquitous and occurred in varying, but always significant, concentrations in almost all nerve cells. Isoform 2 was abundant in cerebellar Purkinje cells but less concentrated in other brain regions. Isoform 3 had a predominantly extra neuronal location, e.g. it was abundant in the choroid plexuses. The three isoforms were found to be distributed in a highly characteristic manner, suggesting that nerve cells have different requirements for the preservation of their intracellular calcium homeostasis.

Animals↗
PubMed1997

Plasma membrane calcium pump: structure, function and relationships.

The plasma membrane Ca-pump (134 kDa) is stimulated by calmodulin and by other treatments (exposure to acidic phospholipids, treatments with proteases, phosphorylation by protein kinases A or C, self-association to form oligomers). It is the product of four genes (in humans), but additional isoforms originate through alternative mRNA spicing. Most of the pump mass protrudes into the cytoplasm with three main units. The calmodulin binding domain is located in the C-terminal protruding unit. The domain is a positively charged segment of about 25 residues. The calcium-activated protease calpain activates the pump by removing its calmodulin binding domain and the portion C-terminal to it. The-resulting 124 KDa fragment has been used to test the suggestion of an autoinhibitory function of the calmodulin binding domain. The latter interacts with two domains of the pump, one located close to the active site in the mid-cytoplasmic protruding unit, the other in the first (N-terminal) protruding unit. The isoforms of the pump show variations in the regulatory domains, e.g., alternative mRNA splicing can eliminate the domain phosphorylated by protein kinase A, or alter the sensitivity of the pump to calmodulin. This occurs by inserting sequences rich in His between calmodulin binding subdomains A and B. The inserted domain(s) confer pH sensitivity to the binding of calmodulin. Calcium binding sites have been found in acidic regions preceding and following the calmodulin binding domain.

Animals↗
PubMed1997

Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells.

The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.

Calcium↗
PubMed1997

Calcium signalling in the cell nucleus.

The First European Conference on Calcium Signalling in the Cell Nucleus took place in Baia Paraelios, Calabria, Italy, from 4-8 October 1997. It was organized by O. Bachs, E. Carafoli, P. Nicotera and L. Santella (local organizers, G. Bagetta and D. Rotiroti) and attended by about 90 specialists. The scientific content was very high and the discussions were particularly intense. Considering that the area is famous for its controversies, one could perhaps have expected aggressive overtones. Instead, in spite of the liveliness of the discussions, the atmosphere was congenial and constructive. The controversies have not disappeared, but a better understanding of some of their origins is now well under way.

Calcium↗
PubMed1997

Calcium signaling in the cell nucleus.

Regulation of Ca2+ in the nucleus is a debated issue, essentially due to the presence in the envelope of the pores, which are large enough to permit the passive traffic of small molecules like Ca2+. Work with a number of cell systems has shown that Ca2+ diffuses freely in and out of the nucleus, whereas other studies have suggested instead that the nuclear envelope could become an efficient Ca2+ filter: electrophysiological work has shown that it could become impermeable to ions, and persistent nucleus cytoplasmic Ca2+ gradients have been documented in various cell types. The problem of the control of nuclear Ca2+ thus is still open: mechanisms for gating of the pores, based on the state of depletion of the cell Ca2+ stores, have been proposed. Irrespective of the mechanisms for possible pore gating, a final picture on the traffic of Ca2+ in and out of the nucleus must also include the Ca2+ pump as well as the InsP3 and cyclic ADP ribose-modulated Ca2+ channels in the envelope. The channels can be activated by their ligands from inside the nucleus, producing Ca2+ transients in the nucleoplasm; the machinery for producing InsP3 has been documented in the envelope. Most Ca2+-sensitive nuclear functions are jointly modulated by Ca2+ and calmodulin: calmodulin-dependent kinases and the calmodulin-dependent phosphatase calcineurin have been documented in the nucleus. An interesting case for the modulation of intranuclear processes by calmodulin-dependent kinases is that of immediate early genes, i.e., CREB. Other Ca2+-modulated nuclear processes are calmodulin independent: chief among them is the intranucleosomal cleavage of chromatin and the fragmentation of nuclear proteins during apoptosis.

Animals↗
PubMed1996

The plasma membrane calcium pump: recent developments and future perspectives.

The Ca2+ pump of the plasma membrane (PMCA) is regulated by a number of agents. The most important is calmodulin (CaM), which binds to a domain located in the C-terminal portion of the pump, removing it from an autoinhibitory site next to the active site. The CaM-binding domain is preceded by an acidic sequence which contains a hidden signal for endoplasmic reticulum (ER) retention. Chimeras of the PMCA and endoplasmic reticulum (SERCA) pumps have revealed the presence of a strong signal for ER retention in the first 45 residues of the SERCA pump. Four gene products of the PMCA pump are known: two of them (1 and 4) are ubiquitously expressed, two (2 and 3) are specific for nerve cells and may be induced by their activation. Mutagenesis work has identified four residues in three of the transmembrane domains of the pump which may be components of the trans-protein Ca2+ path. The mutation of two of these residues alters the membrane targeting of the pump.

Amino Acid Sequence↗
PubMed1996

Purification of active calpain by affinity chromatography on an immobilized peptide inhibitor.

Most purification schemes of calpain (CANP) involve a number of chromatographic steps. The final preparations often contain impurities, including degradation fragments. Two peptide-affinity columns were developed, using peptides of 27 amino acids and 30 amino acids, corresponding to the products of exons 1B and 1C, respectively, of the natural inhibitor (calpastatin) gene, coupled to CNBr-activated Sepharose 4B. Crude preparations of calpain, isolated by anion-exchange chromatography on a DEAE-Sepharose column, were incubated with a reversible or an irreversible synthetic inhibitor which blocks the catalytic subunit of the enzyme in the inactive 80-kDa form. The crude preparation was then loaded onto the peptide column in the presence of calcium. Calpain was eluted with an EGTA-containing buffer. Using the two peptide-affinity columns connected in tandem, calpain was isolated with a high degree of purity, suitable for structural and mechanistic studies, i.e. as an 80/30-kDa heterodimer or in the form of dissociated monomers.

Amino Acid Sequence↗
PubMed1996

The organization of the human gene NCX1 encoding the sodium-calcium exchanger.

The intron-exon organization of the human Na/Ca-exchanger gene NCX1 and of the N-terminal half of the related gene NCX2 has been determined. The NCX1 gene consists of 12 exons spread over 200 kb on chromosome 2 close to STS D2S2328 and encodes a 6.2-kb transcript. Both NCX1 and NCX2 feature an unusual 1.8-kb exon, containing two-thirds of the protein coding sequence and a similar area of the coding sequences split into several small exons, displaying tissue-specific alternative splicing. The similar intron positions in the "cardiac" (NCX1) and "brain" (NCX2) mammalian exchanger genes suggest their origin from the recent duplication and translocation of a common ancestral gene, a putative precursor of which has been identified in the nematode Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed1996

Expression of an active Na+/Ca2+ exchanger isoform lacking the six C-terminal transmembrane segments.

The short isoform of the Na+/Ca2+ exchanger (67 kDa) that is produced by alternative splicing during the expression of the 6 kb canine exchanger cDNA in 293 cells was separately expressed in the same system. The protein consisted of the five N-terminal transmembrane segments and of a large portion of the main hydrophilic loop, but lacked the six C-terminal hydrophobic segments of the regular protein (108 kDa). Very high RNA levels were found after transient cell transfection with plasmid DNA encoding this truncated isoform. The RNA processing, the translation and targeting of the resulting protein to the plasma membrane appeared to be less efficient than those of the 108-kDa polypeptide produced in the same system. The Na(+)-dependent Ca(2+)-uptake activity of 293 cells expressing the short isoform was measured by an isotopic rapid filtration method, whereas the current associated with Ca2+ extrusion was measured in electrophysiological patch-clamp experiments. The results showed that the expressed isoform functioned in the typical reverse and forward Na+/Ca2+ exchange modes. In both the electrophysiological and the isotopic measurements the activity of the short isoform was 6-7-fold lower than that of the 108-kDa protein expressed in the same system. However, lower amounts of the short isoform reached the plasma membrane: its specific activity could thus be significantly higher. Possibly, the short isoform could form a dimer in which a second 67 kDa polypeptide replaces the C-terminal part of the 108-kDa protein.

Alternative Splicing↗
PubMed1996

Expression and functional characterization of isoforms 4 of the plasma membrane calcium pump.

PMCA isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII had a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but had a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone was almost 3 times higher than in PMCA4CI and was increased by La3+ less than in the PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) had no Ca2+-dependent ATPase activity, but was still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform was retained in the endoplasmic reticulum; changes in membrane architecture apparently occurred during its expression; the C-terminal portion of the isoform was located in the cytosol, indicating that the deletion of the 10th transmembrane domain resulted in the loss of at least another transmembrane domain.

Alternative Splicing↗
PubMed1996

Colocalization of the dihydropyridine receptor, the plasma-membrane calcium ATPase isoform 1 and the sodium/calcium exchanger to the junctional-membrane domain of transverse tubules of rabbit skeletal muscle.

The subcellular distribution of the calmodulin-stimulated plasma-membrane Ca(2+)-ATPase (PMCA) has been studied in rat and rabbit skeletal muscle cells by indirect (calmodulin gel overlays) and direct (Western blotting with specific antibodies) methods. It has also been studied in situ in immunocytochemistry experiments. The distribution of PMCA has been compared with that of the NA+/Ca2+ exchanger and of the dihydropyridine receptor, which has been studied by Western blotting with specific antibodies. Both PMCA and the Na+/Ca2+ exchanger had a dual localization, i.e., they were found in the plasma membrane and in the transverse-tubule fractions of the two main types of skeletal muscles studied. The pump and the exchanger were not diffusely distributed in the transverse-tubule-membrane system, but specifically confined to the membrane domain where the dihydropyridine receptor was also localized, i.e., the junctional membrane. Experiments with isoform-specific antibodies have shown that the pump isoform expressed in skeletal muscle is PMCA 1.

Animals↗
PubMed1996

Molecular biological studies of the cardiac sodium-calcium exchanger.

The intron-exon organization of the entire human Na-Ca-exchanger gene NCX1 and of the central part of the related gene NCX2 has been determined. The NCX1 gene is at least 75 kb long and consists of at least 12 exons, the two largest (the 2nd and the 12th) coding for the N-terminal half of the exchanger sequence and for the last three C-terminal transmembrane domains. They also code for the 3.3-kb 3'-untranslated region and account for more than 90% of the length of the mature mRNA. The remainder of the NCX1 (NCX2) gene, coding for a putative cytoplasmic regulatory domain, is split into 9 (7) small exons. In spite of the limited (65%) average homology of the two cDNAs, analogous exons are readily identified within this portion of the two genes based on their high (80-95%) pairwise homology and similar patterns of differential splicing in brain. Human YAC clones have been identified in the CEPH library, which contain the entire NCX1/2 and NCKX1 (retinal rod exchanger) genes, and are used for chromosomal localization of the three genes. A distant homolog of the mammalian NCX genes has been identified in the C. elegans EST database and has been completely sequenced. It encodes a 20% shorter protein, which has an average 55% homology to human NCX1, and lacks most of the region that is known to be encoded by multiple differentially spliced exons in vertebrates. Comparison of available data on the gene structure of the NCX homologs in various species suggests that this protein has emerged in the primitive nervous system and has been subsequently adapted to other cellular environments by the use of novel domains, encoded in additional exons.

Amino Acid Sequence↗
PubMed1996

Expression of Na(+)-Ca2+ exchanger with modified C-terminal hydrophobic domains and enhanced activity.

A 6-Kb canine cDNA fragment complementary to the 5' region of the 7-Kb mRNA encoding the cardiac Na+-Ca2+ exchanger was expressed in human kidney 293 cells. The mRNA products were reverse transcribed and amplified by PCR. The determined DNA sequence of the amplified DNA fragments revealed the presence of an intron that was alternatively spliced. The partial exon sequence, located at the 3' end of the 6-Kb cDNA, was alternatively connected to bases 3198, 2821, 2620 and 1844 in four types of splicing products identified. In the largest product the adjoining exon was located after the putative stop codon of the regular sequence. In a second and third type of shortened transcripts, a hydrophobic sequence encoded by the spliced-in exon was linked with the 4th or the 5th extracellular loops, and could possibly replace transmembrane segments 9 or 11. In the fourth type of spliced transcript the in-frame exon sequence introduced one Leu followed by a stop codon in the large hydrophilic loop. Measurements of Ca2+ uptake in 293 cells expressing the modified exchanger indicated a higher activity in comparison with 293 cells expressing the 3.7-Kb cDNA, in which this alternative splicing does not occur. Deletion mutagenesis of the C-terminal region encoded by the spliced-in exon was performed to investigate its role in the enhancement of the transport activity.

Alternative Splicing↗
PubMed1996

Mutation of conserved residues in transmembrane domains 4,6 and 8 causes loss of Ca2+ transport by the plasma membrane Ca2+ pump.

Mutants of the plasma membrane Ca2+ pump (PMCA), in which amino acids in transmembrane domains (TM) 4, 6, and 8 had been replaced, have been expressed in COS-7 cells. They were analyzed functionally by measuring the uptake of Ca2+ in microsomal preparations and by following the formation of the phosphorylated intermediate from ATP and from phosphate. The mutated residues corresponded to amino acids whose mutation in the sarcoplasmic reticulum pump (SERCA) caused loss of Ca2+ transport by the pump protein: however, only four of the six SERCA residues were conserved in the PMCA pump. Mutation of Glu423 (TM4), Asn879 or Asp883 (TM6), or Gln97l (TM8) suppressed Ca2+ transport by the pump and its ability to form the phosphorylated intermediate starting from ATP. By contrast, the ability of these mutants to form the intermediate starting from phosphate was not impaired. In two mutants (Glu423 and Asp883) it was even enhanced. Two conserved Pro residues of TM4 were also mutated, leading to the loss of the ability of the pump to form the Ca2+- and ATP-dependent phosphorylated intermediate. Unexpectedly, two of the mutations (Asn879 and Gln971) led to the mistargeting of the mutated proteins, i.e., to their retention in the endoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1996

Detection of a new polymorphism in the plasma-membrane Ca2+ ATPase isoform-3 gene and its exclusion as a candidate for X-linked myotubular myopathy (MTM1).

The severe neonatal centronuclear/myotubular myopathy (XLMTM) is an X-linked disorder characterized by generalized muscle weakness, hypotonia and serious respiratory insufficiency. The gene for this disease has been assigned to the long arm of chromosome X in the Xq28 band. Ca2+ ATPase isoform-3 (ATP2B3) has also been mapped to the human Xq28 region. Moreover, it is expressed in fetal but not in adult muscle, suggesting the developmental regulation of gene transcription. These findings render the ATP2B3 gene as an interesting candidate gene for XLMTM. Four families and 7 unrelated XLMTM patients have been analysed by using cDNA and genomic probes of ATP2B3. No large deletions or duplications have been found but a new EcoRI polymorphism has been identified. In addition, the DNA of an XLMTM male deletion patient has been hybridized with the ATP2B3 gene sequences. Our results therefore support the exclusion of ATP2B3 as the causal disease gene of XLMTM. The isolation of the MTM1 gene has recently been reported by another group. However, our approach has led to the detection of a new polymorphism that is an informative marker for linkage and mutation studies in other Xq28-mapped neurological or neuromuscular disorders.

Adult↗
PubMed1996

Binding of cytosolic proteins to myofibrils in ischemic rat hearts.

Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.

Amino Acid Sequence↗