Calcium signalling in health and disease

Topography of the PMCA pump with the interactors known so far.

Topography of the PMCA pump with the interactors known so far.
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Calcium is essential for the stability of bones, but it is also a very important carrier of biological signals. It regulates cell life from its origin at fertilization to its end in the apoptotic process. In between, it controls important cell functions, among them motility, secretion, (metabolic) production of fuels, gene transcription. The complexity of the signaling function demands the precise regulation of Ca²⁺. This is accomplished through the reversible binding to specific proteins, but especially through its transport across cell membranes. As long as Ca²⁺ is kept under careful control cells function correctly, making use of its messenger function. But if the control of the concentration and movements of Ca²⁺ fails, damage of various degrees of severity, up to cell death, ensues. In the last two years, the work of the Group has analyzed the cellular homeostasis of Ca²⁺, under conditions of normal cell life as well as in cells experiencing various forms of pathology. The emphasis has been on membrane Ca²⁺ transporters, and on neurons as the cell type.

Protein partners for the plasma membrane Ca²⁺ transporters

Two systems export to the external spaces the Ca²⁺ that has entered cells through specific channels: a high affinity Ca-ATPase (PMCA pump) and a low affinity Na/Ca exchanger (NCX). Higher eucaryotic cells contain 4 basic isoforms of th pump. Two are expressed ubiquitously, two only in the nervous system. One possible explanation for the existence of so many pump variants is the isoform-specific interaction with (regulatory) protein partners. Figure 1 shows the pump interactors kwown so far from work that has not considered isoform specificity. We have found that ubiquitous PMCA4 interacts with a protein (14,3,3) that is now attracting great attention. The interaction, which occurs at the N-terminal portion of the pump, is inhibitory. Tissue-specific PMCA2 instead does not interact. The isoform specific interaction suggests a novel mechanism for the regulation of the activity of PMCA pumps. Extension of the analysis to the NCXs, has shown that they also interact with protein 14.3.3. The interaction is inhibitory, and it occurs with all 3 NCX isoforms.

Transcriptional regulation of NCX by the gene silencer DREAM

Autoregulation is a distinct property of the Ca²⁺ signal. We have studied the transcriptional repression of a NCX isoforms (NCX3), which is very important to Ca²⁺ homeostasis in neurons, by a gene silencer (acronym DREAM). This is a Ca²⁺ binding protein that binds to DNA sites in the promoter of the NCX3 gene, blocking it until the binding of Ca²⁺ removes it from the DNA, reactivating transcription. The repression is specific for NCX3. If the Ca²⁺ binding motifs of DREAM are mutated, the silencing of the gene becomes permanent. Cerebellar granule neurons from transgenic mice with mutated DREAM have decreased viability when exposed to stresses, e.g. the increased influx of Ca²⁺. The finding shows that DREAM may regulate the viability of these neurons by modulating the Ca²⁺ extruding ability of NCX3. In a related project we studied the homeostasis of Ca²⁺ in the organelles of stressed cerebellar granules, and have developed a virus-mediated system to transfect very efficiently the granules with the Ca²⁺ indicator aequorin (Figure 2).

The sodium /calcium exchanger in the process of neuronal degeneration

Altered cellular Ca²⁺ homeostasis in neuronal death is a long-standing interest of our Group. We have studied the role of NCX3 as a target of the Ca²⁺ dependent protease calpain in the death of cerebellar granule neurons. Ca²⁺ has been transiently by activating its influx with glutamate. The increased Ca²⁺ activates calpain, which cleaves NCX3 causing Ca²⁺ overload and decreasing cell viability. Cell viability is restored to normal by blocking calpain activity. These findings reinforce the conclusion from the DREAM project that NCX3 is essential for the control of Ca²⁺ homeostasis in neurons.

Hereditary deafness caused by defects in the PMCA pump (with F. Mammano)

Some forms of hereditary deafness are caused by mutations in the gene of PMCA2, which is expressed in the stereocilia of the hair cells of the Corti organ. The PMCA variant expressed there is doubly spliced, at site A (N terminal) and site C (C terminal). We have cloned and expressed it in model cells, and shown, (aequorin was the recombinant Ca²⁺ probe) that it does not react with rapid activation to the arrival of a Ca²⁺ pulse. However, it has a very high non-activated pumping activity. We have analyzed two mice mutants (one of them novel) and a new human mutation, and have found depressed pumping activity of the PMCA2. Evidently, the defect disrupts the balance of Ca²⁺ between stereocilia and endolymph, disturbing the mechano-transduction currents that transmit the sound stimuli to the brain.

Altered calcium homeostasis in striatal neurons in Huntington's disease (HD)

The disease damages striatal spiny neurons and has been suggested to be related to disturbed Ca²⁺ homeostasis. We have studied striatal neurons from wt mice and from mice carrying the Q extension in huntingtin typical of HD, and have found striking alterations in the enzymes/receptor/channels that control Ca²⁺. Of the two plasma membrane receptors that regulate it in these neurons, the purinergic receptors are strongly down-regulated (RT-PCR), the bradykinin receptors are up-regulated in HD neurons. The rate-limiting step of the InsP3 cycle (the IP1 phosphatase) is strongly down-regulated, as is the InsP3 channel itself, while the phospholipase C that produces InsP3 is upregulated. Evidently, HD induces a re-programming of the expression of the components of the InsP3 cycle. The end-result of it is a slowed production of InsP3 when HD neurons are stimulated with the two receptor agonists above.

Synoptic CV

Honours

** Selected Publications (VIMM)

Selected Seminars

Contact

Ernesto Carafoli Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 240 Tel. lab.:  (+39) 049 7923 241

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