Laura Fedrizzi

The plasma membrane Ca2+ ATPase of animal cells : structure, function and regulation

Most important processes in cell life are regulated by calcium (Ca2+). A number of mechanisms have thus been developed to maintain the concentration of free Ca2+ inside cells at the level (100-200nM) necessary for the optimal operation of the targets of its regulatory function. The systems that move Ca2+ back and forth across membranes are important actors in its control. The plasma membrane calcium ATPase (PMCA pump) which ejects Ca2+ from all eukaryotic cell types will be the topic of this contribution. The pump uses a molecule of ATP to transport one molecule of Ca2+ from the cytosol to the external environment. It is a P-type ATPase encoded by four genes (ATP2B1-4), the transcripts of which undergo different types of alternative splicing. Many pump variants thus exist. Their multiplicity is best explained by the specific Ca2+ demands in different cell types. In keeping with these demands, the isoforms are differently expressed in tissues and cell types and have differential Ca2+ extruding properties. At very low Ca2+ concentrations the PMCAs are nearly inactive. They must be activated by calmodulin, by acid phospholipids, by protein kinases, and by other means, e.g., a dimerization process. Other proteins interact with the PMCAs (i.e., MAGUK and NHERF at the PDZ domain and calcineurin A in the main intracellular domain) to sort them to specific regions of the cell membrane or to regulate their function. In some cases the interaction is isoform, or even splice variant specific. PMCAs knock out (KO) mice have been generated and have contributed information on the importance of PMCAs to cells and organisms. So far, only one human genetic disease, hearing loss, has been traced back to a PMCA defect.

Calcium Homeostasis and Mitochondrial Dysfunction in Striatal Neurons of Huntington Disease

Dysfunctions of Ca2+ homeostasis and of mitochondria have been studied in immortalized striatal cells from a commonly used Huntington disease mouse model. Transcriptional changes in the components of the phosphatidylinositol cycle and in the receptors for myo-inositol trisphosphate-linked agonists have been found in the cells and in the striatum of the parent Huntington disease mouse. The overall result of the changes is to delay myo-inositol trisphosphate production and to decrease basal Ca2+ in mutant cells. When tested directly, mitochondria in mutant cells behave nearly normally, but are unable to handle large Ca2+ loads. This appears to be due to the increased Ca2+ sensitivity of the permeability transition pore, which dissipates the membrane potential, prompting the release of accumulated Ca2+. Harmful reactive oxygen species, which are produced by defective mitochondria and may in turn stress them, increase in mutant cells, particularly if the damage to mitochondria is artificially exacerbated, for instance with complex II inhibitors. Mitochondria in mutant cells are thus peculiarly vulnerable to stresses induced by Ca2+ and reactive oxygen species. The observed decrease of cell Ca2+ could be a compensatory attempt to prevent the Ca2+ stress that would irreversibly damage mitochondria and eventually lead to cell death.

A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.

Functional specificity of PMCA isoforms

Abstract:  In mammals, four different genes encode four PMCA isoforms. PMCA1 and PMCA4 are expressed ubiquitously. PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of such elevated number of isoforms is not clear, but it would be plausible to relate it to the cell‐specific demands of Ca2+ homeostasis. To characterize functional specificity of PMCA variants we have investigated two aspects: the effects of the overexpression of the different PMCA variants on cellular Ca2+ handling and the existence of possible isoform‐specific interactions with partner proteins using a yeast two‐hybrid technique. The four basic PMCA isoforms were coexpressed in CHO cells together with the Ca2+‐sensitive recombinant photoprotein aequorin. The effects of their overexpression on Ca2+ homeostasis were monitored in the living cells. They had revealed that the ubiquitous isoforms 1 and 4 are less effective in reducing the Ca2+ peaks generated by cell stimulation as compared to the neuron‐specific isoforms 2 and 3. To establish whether these differences were related to different and new physiological regulators of the pump, the 90 N‐terminal residues of PMCA2 and PMCA4 have been used as baits for the search of molecular partners. Screening of a human brain cDNA library with the PMCA4 bait specified the ɛ‐isoform of protein 14‐3‐3, whereas no 14‐3‐3 ɛ clone was obtained with the PMCA2 bait. Overexpression of PMCA4/14‐3‐3 ɛ (but not of PMCA2/14‐3‐3 ɛ) in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired. Thus, the interaction with 14‐3‐3 ɛ inhibited PMCA4 but not PMCA2. The role of PMCA2 has been further characterized by Ca2+ measurements in cells overexpressing different splicing variants. The results indicated that the combination of alternative splicing at two different sites in the pump structure was responsible for different functional characteristics of the pumps.

Interplay of the Ca2+-binding Protein DREAM with Presenilin in Neuronal Ca2+ Signaling

The Ca2+-binding protein DREAM regulates gene transcription and Kv potassium channels in neurons but has also been claimed to interact with presenilins, which are involved in the generation of β-amyloid and in the regulation of the Ca2+ content in the endoplasmic reticulum. The role of DREAM in Ca2+ homeostasis was thus explored in SH-SY5Y cells stably or transiently overexpressing DREAM or a Ca2+-insensitive mutant of it. The overexpression of DREAM had transcriptional and post-transcriptional effects. Endoplasmic reticulum Ca2+ and capacitative Ca2+ influx were reduced in stably expressing cells. The previously shown down-regulation of Na+/Ca2+ exchanger 3 expression was confirmed; it could cause a local increase of subplasma membrane Ca2+ and thus inhibit capacitative Ca2+ influx. DREAM up-regulated the expression of the inositol 1,4,5-trisphosphate receptor and could thus increase the unstimulated release of Ca2+ through it. The transient coexpression of DREAM and presenilin potentiated the decrease of endoplasmic reticulum Ca2+ observed in presenilin-overexpressing cells. This could be due to a direct effect of DREAM on presenilin as the two proteins interacted in a Ca2+-independent fashion.

Calcium and signal transduction

Cell signaling is an essential process in which a variety of external signals, defined as first messengers, are translated inside the cells into specific responses, which are mediated by a less numerous group of second messengers. The exchange of signals became a necessity when the transition from monocellular to pluricellular life brought with it the division of labor among the cells of the organisms: unicellular organisms do not depend on the mutual exchange of signals, as they essentially only compete with each other for nutrients. Calcium (Ca2+) was selected during evolution as second messenger, because its chemistry made it a much more flexible ligand than the other abundant cations in the primordial environment (Na+, K+, Mg2+). Ca2+ can accept binding sites of irregular geometries and is thus ideally suited to be a carrier of biological information. The Ca2+ signal has properties that set it apart from those of all other biological messengers: they will be reviewed in this contribution. Among them, the ambivalent character of the Ca2+ signal is the most important: while essential to the viability of the cells, it can also easily become a conveyor of doom.

Plasma-membrane calcium pumps and hereditary deafness.

In mammals, four different genes encode four PMCA (plasma-membrane Ca(2+)-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca(2+) homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca(2+) control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps.

Beauty and its perception: historical development of concepts, neuroaesthetics, and gender-differences

Perception, appreciation, preference, pleasure, emotion, truth, interpretation: these are only few of the concepts we encounter when we try to understand the intrinsic meaning of beauty. The secret of the sensations we feel in front of something beautiful has accompanied us since the origin of humankind, when our first ancestors with sufficient cognitive abilities understood and appreciated beauty. The representation of beauty occurs through the production first, and the fruition then, of the works of art. We have many useful instruments to approach the understanding of what beauty really is: from the philosophical theories of aesthetics, to the evolutionary influence on our system of perception and production of art works, to the tools of neuroaesthetics, which explore the brain structures and the processes which mediate the aesthetic experience. In addition, the neuroanatomical studies on the gender differences in the perception of beauty, and their surprising details enhance and refine our comprehension of aesthetics.

Ca2+-activated Nucleotidase 1, a Novel Target Gene for the Transcriptional Repressor DREAM (Downstream Regulatory Element Antagonist Modulator), Is Involved in Protein Folding and Degradation

Background: Ca2+-activated nucleotidase 1 (CANT1), an Endoplasmic Reticulum-Golgi resident nucleoside diphosphatase may have a role in protein quality control. Results: CANT1 has been identified as a novel target of the Ca2+-dependent transcriptional repressor DREAM. Conclusion: CANT1 down-regulation increased protein degradation. CANT1 overexpression enhances Ca2+ levels in Golgi apparatus, indicating a role in Ca2+ homeostasis. Significance: Protein degradation represents a novel process modulated by DREAM. DREAM is a Ca2+-dependent transcriptional repressor highly expressed in neuronal cells. A number of genes have already been identified as the target of its regulation. Targeted analysis performed on cerebella from transgenic mice expressing a dominant active DREAM mutant (daDREAM) showed a drastic reduction of the amount of transcript of Ca2+-activated nucleotidase 1 (CANT1), an endoplasmic reticulum (ER)-Golgi resident Ca2+-dependent nucleoside diphosphatase that has been suggested to have a role in glucosylation reactions related to the quality control of proteins in the ER and the Golgi apparatus. CANT1 down-regulation was also found in neuroblastoma SH-SY5Y cells stably overexpressing wild type (wt) DREAM or daDREAM, thus providing a simple cell model to investigate the protein maturation pathway. Pulse-chase experiments demonstrated that the down-regulation of CANT1 is associated with reduced protein secretion and increased degradation rates. Importantly, overexpression of wtDREAM or daDREAM augmented the expression of the EDEM1 gene, which encodes a key component of the ER-associated degradation pathway, suggesting an alternative pathway to enhanced protein degradation. Restoring CANT1 levels in neuroblastoma clones recovered the phenotype, thus confirming a key role of CANT1, and of the regulation of its gene by DREAM, in the control of protein synthesis and degradation.

Reduced Mid1 Expression and Delayed Neuromotor Development in daDREAM Transgenic Mice

Downstream regulatory element antagonist modulator (DREAM) is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. Previous work has shown a role for DREAM in cerebellar function regulating the expression of the sodium/calcium exchanger 3 (NCX3) in cerebellar granular neurons to control Ca2+ homeostasis and survival of these neurons. To achieve a global view of the genes regulated by DREAM in the cerebellum, we performed a genome-wide analysis in transgenic cerebellum expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Here we show that DREAM regulates the expression of the midline 1 (Mid1) gene early after birth. As a consequence, daDREAM mice exhibit a significant shortening of the rostro-caudal axis of the cerebellum and a delay in neuromotor development early after birth. Our results indicate a role for DREAM in cerebellar function.