Browsing by Subject "Calcium Channels"
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Item Ca2+ Signaling in the Near Plasma Membrane Microdomain of Non-Excitable Cells(2011-02-01T19:37:03Z) Yao, Jian; Bezprozvanny, IlyaCalcium is the most versatile second messenger and plays fundamental roles in orchestrating enzyme secretion in exocrine acinar cells. Previous studies in excitable cells demonstrated the existence of high Ca2+ microdomains. The major function of such microdomains is to create high local calcium concentration to activate various calcium-dependent signaling events. However, in non-excitable cells, direct evidence of such microdomains is absent. The goal of my study is to characterize the properties of high Ca2+ microdomains in acinar cells and explore its physiological relevance in the context of the secretory functions. By combining Total Internal Reflection Fluorescence Microscopy (TIRFM) technique and wide-field fluorescence imaging, I was able to quantify and compare changes in the concentration of free Ca2+ in the near membrane microdomains (?[Ca2+]PM) and in the bulk cytosol (?[Ca2+]Cyto). ?[Ca2+]PM is about 3-fold larger than ?[Ca2+]Cyto under maximal agonist stimulation, while resting [Ca2+]PM and [Ca2+]Cyto shows no difference. Near membrane microdomains also showed greater Ca2+ influx following store depletion induced either by activating surface receptor or by inhibiting SERCA pump. In response to physiological strength of stimulation, Ca2+ oscillation in the two compartments showed significantly different dynamics. The activation mechanisms of the Ca2+-induced Ca2+ release (CICR) are well established in cardiac and skeletal muscles and involves high Ca2+ microdomains. My study was the first to demonstrate the presence of CICR in the parotid acinar cell. In these cells, minimal activation of Ca2+ influx by partially depleting the stores, either by directly activating the cell surface receptor or by inhibiting SERCA, leads to an explosive release of Ca2+ from the majority of the stores, mediated presumably by RyR away from microdomains. The last part of my study is on the effects of chronic ER stress on Ca2+ signaling. The study suggests that ER stress induced by PERK mutation impeded both the efficiency and fidelity of Ca2+ signaling. My work validates the existence of near plasma membrane microdomains in non-excitable exocrine cells. The fact that [Ca2+]PM and [Ca2+]Cyto differ in many ways suggests that microdomains is the central signaling platform in these cells.Item Modulating Calcium Signaling Pathways in Cerebellar Purkinje Cells Alleviates Spinocerebellar Ataxia 2(2012-07-16) Kasumu, Adebimpe Wakila; Bezprozvanny, IlyaSpinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder characterized by progressive ataxia. SCA2 results from the polyglutamine expansion in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PC) are primarily affected in SCA2, but the cause of PC dysfunction, PC death and motor incoordination in SCA2 is poorly understood. It has been reported that mutant, but not wild type Atx2, specifically binds to the inositol 1,4,5-trisphosphate receptor (InsP3R) and increases its sensitivity to activation by IP3. Thus, this toxic gain-of-function of Atx2 results in supranormal calcium (Ca2+) release from the PC endoplasmic reticulum and may play a key role in the development of SCA2 pathology. The primary focus of this dissertation will be to further elucidate the underlying mechanism of SCA2 pathogenesis, identify therapeutic targets and develop a potential treatment of SCA2. The first part of this dissertation will test the hypothesis that suppressing InsP3R-mediated Ca2+ signaling alleviates age-dependent dysfunction, and degeneration of PCs in SCA2 mice. The second part of this dissertation will focus on testing the efficacy of novel compounds that modulate calcium-activated potassium (SK) channels in the symptomatic treatment of SCA2. I conclude from this work that supranormal InsP3--Ca2+ signaling plays an important role in SCA2 pathogenesis. Partial inhibition of InsP3-mediated Ca2+ signaling or regularizing PC firing with SK channel modulators could provide therapeutic benefit for the patients afflicted with SCA2 and possibly other SCAs. [Keywords: calcium, Spinocerebellar, Purkinje, ataxia, potassium]Item Role of the Vitamin D Receptor in Insulin Secretion and Beta Cell Function(2012-07-20) Kjalarsdottir, Lilja; Repa, Joyce1,25-dihydroxyvitamin D3 (VitD) is a ligand for the Vitamin D Receptor (VDR, NR1I1), which is a member of the family of Nuclear Hormone Receptors (NHR). Previously, the Repa lab identified VDR as the fourth most abundant NHR in mouse islets based on mRNA levels, also, VDR is clearly present in human islets [1]. In the past years multiple epidemiological studies have implicated Vitamin D deficiency in the development of Type 2 Diabetes, however no reports have described any mechanism(s) linking VitD status with pancreatic islet function. Therefore, my studies have focused on the role of Vitamin D and VDR in islet biology. Preincubation of isolated mouse and human islets with Vitamin D results in enhanced glucose-stimulated insulin secretion (GSIS). This response is VDR-dependent, as no VitD-mediated change in GSIS is observed in islets obtained from Vdr-null mice. However, VitD causes no changes in gene expression of any of the major islet hormones, nor does it change glucose uptake into primary beta cells. VitD does however increase glucose-stimulated calcium uptake, suggesting that VitD affects transcription of genes involved in calcium flux into the beta cell. To identify molecular mechanisms linking VDR activity to increased insulin secretion and increased glucose-stimulated calcium uptake, we performed global gene expression profiling by microarray in mouse and human islets. These studies identified multiple genes associated with islet function, calcium transport and insulin secretion. One of these genes is the R-type voltage-gated calcium channel, CaV2.3, which is highly upregulated by VitD in human and mouse islets. We identified a strong VDR binding element within intron 7 of the Cav2.3 gene that is conserved in mouse and man. With previous reports linking Cav2.3 activity with Type 2 Diabetes, our findings support a role for vitamin D signaling in the regulation of CaV2.3 and calcium uptake to enhance glucose-stimulated insulin secretion by beta cells of the endocrine pancreas. A second VDR target gene we identified in the islet is klotho, a key regulator of phosphate homeostasis. We clearly establish that klotho mRNA and protein are detected in beta cells of mouse islets, at levels sufficient to mediate signal transduction pathways via klotho’s role as a co-receptor for FGF23. By analysis of islets from Klotho-/- mice, we also show that the sialidase activity of klotho may modulate the membrane localization of GLUT-2 to affect glucose-stimulated insulin secretion. In summary, my studies suggest that vitamin D status may impact the beta cell’s capacity to sense glucose levels and respond appropriately to secrete the anabolic hormone, insulin. Future studies involving beta cell-selective deletion of VDR, klotho, and Cav2.3 are now warranted, to elucidate the contribution of islet vitamin D signaling pathways in glucose homeostasis in vivo. The results of studies for my dissertation research provide a needed mechanistic approach, which complements the current clinical and observational reports that exist, regarding potential roles for Vitamin D in the progression of Diabetes. In addition, our identification of numerous Vitamin-D regulated genes of the human and mouse islet can form the basis for future hypothesis-driven research efforts to identify novel therapeutic targets to affect insulin secretion and beta cell function. [Keywords: vitamin D, vitamin D receptor, VDR, islets, diabetes, insulin secretion, microarray, voltage-gated calcium channel, Klotho, bile acids]Item Study of Presynaptic Calcium Channels and a Novel Calcium Channel Calhm1(2010-11-02T18:19:16Z) Ozkan, Emin D.; Bezprozvanny, IlyaCalcium signaling is essential for all cellular processes. In brain these processes include basic synaptic transmission, modification of established synapses, elimination of synapses as well as elimination of whole neurons by way of apoptosis. In this thesis, we have studied two of these processes. One process is synaptic targeting of presynaptic calcium channels. We have tested the hypothesis that synaptic targeting of presynaptic calcium channels depends on carboxy terminal interactions with mint and CASK proteins. To this end we have used mice mutant for P/Q-type calcium channel. Our experiments show that a P/Q-type calcium channel without its carboxy terminal can partially contribute to synaptic transmission. Secondly we have studied the role of Calhm1 gene in modulating calcium signaling pathways. For this purpose we have used overexpression of Calhm1 gene in HEK293 cells as well as in dissociated neuronal cultures. Our experiments show that Calhm1 modulates calcium signaling in HEK293 cell and in certain neurons. Calhm1 overexpression also modulates spontaneous synaptic transmission.