Letteratura scientifica selezionata sul tema "Influx de calcium"

<i>FIG1 </i>is a gene that encodes a transmembrane protein involved in calcium influx a process that is important for stress responses in pathogens fungi such as <i>Candida albicans.</i> A <i>Cafig1 </i>null mutant took up less calcium ions in mating than control strains confirming its role in Ca²⁺ uptake.  Furthermore, <i>C. albicans </i>strains with deletions of <i>FIG1 </i>in other calcium channel mutant backgrounds displayed distinctive phenotypes under vegetative growth conditions, which suggested that Fig1 may be a regulator of other calcium influx systems. Using GFP and <i>LacZ </i>reporter constructs it was shown that in <i>C. albicans FIG1 </i>was induced by mating pheromone and during the interactions of strains of compatible mating type.  Localization of Fig1-GFP studied by con-focal imaging showed that the protein had a peri-nuclear distribution and was also found in the plasma membrane at the tips of shmoos.  The protein appeared to be located within microdomains and the protein sequence was found to contain a putative site for cysteine palmitoylation that may promote such localization. Because the expression of <i>FIG1 </i>was strongly induced during mating, the induction of <i>FIG1 </i>was used to try to detect where mating took place during experimental infection in mice. Surprisingly <i>FIG1 </i>expression could be observed in the murine gut in control inoculations using <i>C. albicans </i>strains that could not undergo mating.  Therefore <i>FIG1 </i>expression is not always strictly mating-dependent.  MAP kinases and calcium-calcineurin signalling pathways, in which Fig1 is involved, were studied using bioinformatics approaches.  It was shown that these signalling pathways contain conserved signalling components taking part in signal transduction via phosphorelay but they had diverged receptors, sensors, effectors, and phosphorelay regulators across different fungal species.

Molecular and Cellular Physiology<br>Ph.D.<br>Pathological cardiac stressors, including persistent hypertension or damage from ischemic heart disease, induce a chronic demand for enhanced contractile performance of the heart. The cytosolic calcium (Ca2+) transient that regulates myocyte contraction must be persistently increased in disease states in order to maintain cardiac output to sustain the metabolic requirements of the body. Associated with this enhanced intracellular Ca2+ ([Ca2+]i) state is pathological cardiac myocyte hypertrophy, which results in large part from the activation of Ca2+-dependent activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling. The puzzling feature of this hypertrophic signaling is that the cytosolic [Ca2+] that controls contractility appears to be separate from the [Ca2+] which activates Cn-NFAT signaling. The overarching theme of this dissertation is to explore the source and spatial constraints of pathological hypertrophic signaling Ca2+ and to investigate how it is possible that sensitive and finely tuned Ca2+-dependent signaling pathways are regulated in the background of massive Ca2+ fluctuations that oscillate between 100nM and upwards of 1-2&#956;M during each cardiac contractile cycle. L-type Ca2+ channels (LTCCs) are a major source of Ca2+ entry in cardiac myocytes and are known to play an integral role in the initiation of myocyte excitation contraction-coupling (EC-coupling). We performed a number of experiments to show that a small population of LTCCs reside outside of EC-coupling domains within caveolin (Cav-3) signaling microdomains where they provide a local source of Ca2+ to activate Cn-NFAT signaling. We designed a Cav-targeted LTCC blocker that could eliminate Cn-NFAT activation but did not reduce myocyte contractility. The activity of Cav-targeted LTCCs could also be upregulated to enhance hypertrophic signaling without affecting contractility. Therefore, we believe that caveolae-localized LTCCs do not participate in EC-coupling, but instead act locally to control the coordinated activation of Cn-NFAT signaling that drives pathological remodeling. Transient Receptor Potential (TRP) channels are also thought to provide a source of Ca2+ for activation of hypertrophic signaling. The canonical family of TRP channels (TRPC) is expressed at low levels in normal adult cardiac tissue, but these channels are upregulated in disease conditions which implicates them as stress response molecules that could potentially provide a platform for hypertrophic Ca2+ signaling. We show evidence that TRPC channel abundance and function increases in cardiac stress conditions, such as myocardial infarction (MI), and that these channels are associated with hypertrophic responses, likely through a Ca2+ microdomain effect. While we found that TRPC channels housed in caveolae membrane microdomains provides a source of [Ca2+] for induction of cardiac hypertrophy, this effect also requires interplay with LTCCs. We also found that TRPC channels have negative effects on cardiac contractility, which we believe are due to local activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) and subsequent modulation of ryanodine receptors (RyRs). Further, we found that inhibiting TRPC channels in a mouse model of MI led to increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling, as well as increased survival. Collectively, the data presented in this dissertation provides comprehensive evidence that Ca2+ regulation of Cn-NFAT signaling and resultant pathological hypertrophy can be coordinated by spatially localized and regulated Ca2+ channels. The compartmentalization of LTCCs and TRPC channels in caveolae membrane microdomains along with pathological hypertrophy signaling effectors makes for an attractive explanation for how Ca2+-dependent signaling pathways are regulated under conditions of continual Ca2+ transients that mediate cardiac contraction during each heart beat. Elucidation of additional Ca2+ signaling microdomains in adult cardiac myocytes will be important in more comprehensively resolving how myocytes differentiate between signaling versus contractile Ca2+.<br>Temple University--Theses

Hyperkalemic periodic paralysis, HyperKPP, is an inherited progressive disorder of the muscles caused by mutations in the voltage gated sodium channel (NaV1.4). The objectives of this thesis were to develop a technique for measurement symptoms in vivo using electromyography (EMG) and to determine the mechanism by which Ca2+ alleviates HyperKPP symptoms, since this is unknown. Increasing extracellular [Ca2+] ([Ca2+]e) from 1.3 to 4 mM did not result in any increases in45Ca2+ influx suggesting no increase in intracellular [Ca2+] ([Ca2+]i) acting on an intracellular signaling pathway or on an ion channel such as the Ca2+sensitive K+ channels. HyperKPP muscles have larger TTX-sensitive22Na+ influx than wild type muscles because of the defective NaV1.4 channels. When [Ca2+] was increased from 1.3 to 4 mM, the abnormal 22Na+ influx was completely abolished. Thus, one mechanism by which Ca2+alleviates HyperKPP symptoms is by reducing the abnormal Na+ influx caused by the mutation in the NaV1.4 channel.

Biochemistry<br>Ph.D.<br>Ca2+ control mechanisms employed by the cell at the plasma membrane include receptor operated, voltage-sensitive, and store operated channels for Ca2+ import. Upon entry into the cytosol, Ca2+ is sequestered by Ca2+ binding proteins, the endoplasmic reticulum (ER), or by mitochondria. The largest Ca2+ store in the cell is the ER where Ca2+ levels approach millimolar levels. The ER regulates cytosolic Ca2+ homeostasis by using Ca2+ binding proteins, the SERCA pump, second messenger Ca2+ release upon IP3 receptor activation, and Ca2+-induced Ca2+ release by ryanodine receptors. Basal cytosolic Ca2+ levels are maintained at around 100nM. The mitochondria begins clearing GPRC-depended cytosolic Ca2+elevation after a short time delay during which the cytosolic Ca2+ concentration exceeds 3&#61549;M. Then, the mitochondria sacrifices a portion of its membrane potential to drive Ca2+ influx across the mitochondrial inner membrane into the matrix. The membrane potential of the mitochondria is created in part by the electron transport, which while transferring electrons, ejects protons from the matrix to the inner membrane space. The rapid mitochondrial Ca2+ uptake decreases mitochondrial membrane potential thus reducing or fully collapsing the mitochondria's ability to generate ATP. This uncoupling of the electron transport chain results in ROS production and decreased cell survival. Mitochondria provide the body with energy that allows a heart to beat, a brain to store memories, and fuels locomotive function. As a stand-alone energy generator, the mitochondria would be interesting, but not dynamic. The dynamic flow of information to the mitochondria through Ca2+ signaling with all the components of symbiotic precision is a true biological phenomenon. In the mitochondria, a complex Ca2+ buffering system of channels, pores, and exchangers directly affects the conversion of chemical potential to ATP. Recent, discoveries of the Ca2+ uniporter (MCU) and other system components have provided the tools to tackle levels of mitochondrion physiologic studies that were not possible only a couple of years ago. There remains a great need for advancement in the understanding of mitochondrial bioenergetics, and undoubtedly, the mitochondria will be viewed as a determinant factor for survival. The mitochondrial inner membrane through its curious construction of 3:1 protein to lipid ratio, carefully regulates the permeability of ions and metabolites. The transport of Ca2+ and other small ions across the inner membrane is an essential signaling pathway for mitochondrial maintenance of metabolic functions, but the mechanisms are still unclear due to a lack of mitochondrial systems biology. For example, the oligomeric MCU with two transmembrane domains is a core component of the major Ca2+ import pathway in mitochondria, and ablation of MCU lowers mitochondrial Ca2+ uptake, however portions such as the highly conserved linker between the two transmembrane was unstudied until recently. Other complex components such as MICU1 and MCUR1, which negatively and positively regulate MCU, are beginning to have their mechanism solved. MICU1 is associated with the mitochondrial inner membrane and has two EF hands, which indicated a possible role in Ca2+ sensing. This role as a Ca2+ sensor proved to be necessary for proper MICU1 inhibition of MCU, but not determinant of MICU1/MCU interaction. MICU1, MCUR1, and MCU are modified in numerous diseases in which a particular component is disproportionately expressed. This is in part due to the classical coupling of gene function to associated transcription factor meaning that because MICU1, MCUR1, and MCU have a Ca2+ flux function, their transcription is also probably controlled by Ca2+ and is altered in chronic inflammation or hypoxic systems such as Ca2+ overload during ischemia/reperfusion. In spite of the low affinity of uniporter, mitochondrial Ca2+ overload occurs due to the close proximity of mitochondria to the ER, however physical tethering of the mitochondria and ER is still not widely accepted. When Ca2+ is physiologically cleared from the cytosol to the mitochondria, it acts as a synchronizing signal to the numerous EF hands present on inner membrane transmembrane proteins and matrix-targeted proteins. . Synchronization of mitochondrial activities is critical for efficiency which has direct implication for both cell growth or damage through the byproduct of inefficiency, mROS (superoxide). Therefore, the EF hands and other Ca2+ response elements enhance the ratio of ATP to superoxide, thus supporting mitogenic function and healthy growth. The inefficient flow of energy leads to dysfunction such as the release of reactive oxygen species (ROS) from the mitochondria. ROS carries its own energy in the chemical form of a radical. This translates into thermodynamically favorable but harmful cellular damage. Sustained import of Ca2+ results in electron transport malfunction followed by loss of membrane potential as seen in ischemia. A common EF hand motif exists on many calcium sensitive proteins. This helix-loop-helix topology recognizes a specific range of calcium concentrations based on the primary and tertiary structure of the domain. Thus, not all EF hands are active at a given physiological Ca2+ concentrations. The Ca2+ is situated in the loop portion by 12 key interactions in a pentagonal bipyramidal geometry. The position of 12th residue supplies two of the interacting oxygen atoms for Ca2+ binding and are conserved as either Glutamate or Aspartate. EF-hand containing proteins do not necessarily transport Ca2+ alone, as many other solutes have also been reported. The EF hand motif can be found on many mitochondrial sensors including LETM-1, MICU1, and non- Ca2+ transporters (Nakayama, Moncrief et al. 1992), suggesting Ca2+ is often the synchronizing signaling molecule but not necessarily transported by the mitochondrial channel of interest. The discovery of the uniporter (MCU) is an exciting event in the field, as many relationships between different transport mechanisms affecting Ca2+ and membrane potential will be elucidated. One such relationship that should be explored is between the uniporter and inorganic phosphate exchange. This relationship may modulate cell death through a critical uptake dynamic between adenine, phosphate and Ca2+ through alternative pathways such as solute carriers. Mitochondrial carriers are crucial for transport across the inner membrane. There are two groups of Ca2+ binding solute carriers in the mitochondria, the aspartate/glutamate carriers (Palmieri, Pardo et al. 2001) and the ATP-magnesium carriers (SCaMC) (Satrustegui, Pardo et al. 2007). Carrier proteins transport molecules by changing shape and therefore can be saturated. Solute carrier activators have been previously reported to include Ca2+, adenosine 3'5'-cyclic monophosphate, protein kinases, and inositol polyphosphates (Dransfield and Aprille 1993). Other previous work has also reported transport of multiple different solutes (Fiermonte, De Leonardis et al. 2004). The higher eukaryote, vertebrate calcium systems, should functionally if not physically interact with conserved lower eukaryote systems such as solute carriers. All known mitochondrial carriers are members of the same family based on three tandem repeats and are predicted to function as oligomers. The human family of these inner mitochondrial membrane proteins is SLC25, and members of the SLC25 family have been identified as the cause of Stanley Syndrome and Amish Microcephaly suggesting the importance of SLC25. SLC25A23 has been proposed to be an ATP-Mg/Pi exchange carrier that allows for both uptake and release of ATP-Mg from mitochondria. As a putative ADP/Pi translocase, it is an interesting component as both ADP and Pi have been shown to play a role in cell survival and cell death. This SLC25A family member is likely to be the critical regulator of these two dynamic molecules. These carriers are stimulated by submicromolar Ca2+ to regulate adenine nucleotide levels in the cytosol and mitochondria. Previous literature has shown SLC25A25 knockout to have little effect on mouse metabolism. SLC25A24 has been shown to be involved in ADP/ATP ratios in the mitochondrial matrix resulting in cytosolic Ca2+ buffering enhancement (21). The functions of SLC25A23 largely remain unknown. It should be pointed out that SLC25A23, SLC25A24, and SLC25A25, Ca2+ induced changes, are not necessarily based on Ca2+ as a channel solute. The ATP/ADP maintained by SLC25 family members may contribute to Ca2+ uptake in the mitochondria and therefore may play a role in cell death through PTP opening. PTP opening is a point of convergence for many cell death pathways. The PTP, which behaves as a voltage-operated channel, can be triggered to open by high mitochondrial Ca2+, ROS, or low membrane potential. In previous studies, SLC25A24 knockdown resulted in increased PTP opening and decreased Ca2+ buffering. Solute carrier family 25 (mitochondrial carrier; phosphate carrier), which includes SLC25A23, SLC25A24, and SLC25A25, transport solutes across the inner membrane, are predicted to form six transmembrane domains sensitive to Ca2+ due to four Ca2+ binding EF hand motifs, and localize to the mitochondria (del Arco and Satrustegui 1998; Iijima, Yamamoto et al. 2001). Based on membrane topology predictions, SLC25 isoforms contain six transmembrane domains with several EF hand motifs. Although the solute carriers in the SCaMC family have been hypothesized to transport adenine, (Aprille 1988) they have never been fully characterized. Mitochondrial solute carriers are found only in eukaryotes (Carafoli and Lehninger 1971; Uribe, Rangel et al. 1992; Palmieri 2004), however Sal1 in yeast has high sequence homology (Kucejova, Li et al. 2008). SLC25A25 knockout was reported to have little effects on mouse metabolism. SLC25A24 has been shown to be involved in ADP/ATP ratios in the mitochondrial matrix resulting in cytosolic Ca2+ buffering enhancement (Traba, Del Arco et al. 2011). The functions of these solute carriers in mitochondrial Ca2+ uptake and mitochondrial ROS are largely unknown.<br>Temple University--Theses