Browsing by Subject "Calcium"
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Item Bariatric surgery and effects on calcium and bone metabolism(2015-06-26) Sakhaee, KhashayarItem Ca2+ Signaling in the Near Plasma Membrane Microdomain of Non-Excitable Cells(2011-02-01) 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 Calcium Triggered Synaptic Vesicle Exocytosis(2007-08-08) Pang, Zhiping; Südhof, Thomas C.Neurotransmitter release is triggered by the action potential induced influx of Ca 2+ into nerve terminals. One of the central questions in neuroscience is how Ca 2+ promotes synaptic vesicles from rest to fusion leading to release of neurotransmitters. In this thesis, I first addressed if synaptogmin-1/SNARE binding is important for synaptic vesicle release. Using two knock-in mouse lines each with single amino-acid substitution, namely D232N and D238N in synaptotagmin-1, combined with electrophysiology, I found evoked release in D232N mutant neuronal cultures is significantly increased, whereas in D238N cultures release is slightly but significantly decreased. Ca 2+ titration curves indicated the apparent Ca 2+-affinity for vesicle release significantly increased in D232N synapses. These data are consistent with biochemical studies that showed that the D232N substitution in synaptotagmin-1 increases Ca 2+-dependent SNARE bindings but leaves phospholipid binding unchanged, whereas the D238N mutant slightly decreased phospholipid binding but leaves SNARE binding insignificantly changed. Second, I addressed if synaptotamgin-2 is another Ca 2+-sensor for synaptic vesicle release. I and my colleagues used two mouse lines: one contains a single amino acid mutation in synaptotagmin-2 (I377N) and one has synaptotagmin-2 ablated from the genome. By using a combination of biophysical, biochemical and functional techniques, we determined that synaptotagmin-2 is a fast synchronous Ca 2+-sensor. Third, in collaboration with Jianyuan Sun, we explored the biophysical properties of the slow Ca 2+-sensor in the Calyx of Held. Using Ca 2+-uncaging combined with electrophysiology, we mapped increasing Ca 2+ concentrations in relation to neurotransmitter release and built a comprehensive mathematical model for the Ca 2+ control of synaptic vesicle fusion. We found compelling evidence for the existence of two Ca 2+- sensors: one (synaptotagmin-2 in the Calyx of Held) is responsible for fast synchronous release, and the other one is responsible for slow delayed synaptic release. Surprisingly, we found the two Ca 2+-sensors have similar apparent Ca 2+ affinities. This study showed clearly that synaptotagmin-2 is a fast Ca 2+-sensor, and gave us a prediction that narrows down the potential candidate for the slow Ca 2+-sensor.Item Dissection of the Roles of Synaptotagmins in Calcium Dependent Neurotransmitter Release(2020-08-01T05:00:00.000Z) Voleti, Sai Rashmi; Nam, Yunsun; Rizo-Rey, José; Chook, Yuh Min; Ross, Elliott M.Neuronal communication is mediated by neurotransmitter release, which is triggered in response to a calcium influx into the presynaptic cell, caused by the arrival of an action potential. Synaptotagmins (Syts) mediate this calcium-dependent regulation of neurotransmitter release by mechanisms that still remain elusive. Syts have two calcium-binding C2 domains (C2A and C2B) which are crucial for their function. Clarifying how Syt C2 domains interact with the neuronal membrane fusion machinery and the membranes is essential for gaining insights into the mechanisms of calcium-triggered neurotransmitter release. To understand the role of Syts in neurotransmitter release, we explored the origins of functional differences between the C2 domains of two Syt isoforms, Syt1 and Syt7, which mediate synchronous and asynchronous release respectively. Calcium binding to the C2A domain is critical for Syt7 functions whereas Syt1 function depends more on calcium binding ability of its C2B domain. By solving the structures of Syt7 C2A and C2AB fragments and by analyzing their intrinsic calcium binding properties by ITC, we showed that these properties do not give rise to the functional differentiation between Syt1 and Syt7. By characterizing the calcium-dependent phospholipid binding of C2 domains by FRET, we demonstrated that C2A and C2B dominate membrane binding in Syt7 and Syt1, respectively. This suggests that membrane affinity of C2 domains might dictate their functional importance for Syt function. In addition to membrane binding, Syt1 C2B domain is also involved in interactions with the SNARE complex, which is the central component of neuronal membrane fusion machinery. Clarifying the molecular details of these interactions is crucial for understanding how Syt1 cooperates with SNARE complex to trigger calcium-dependent membrane fusion. Previous structural studies have shown three distinct SNARE complex-binding interfaces on Syt1 C2B domain, two of which overlap with membrane binding regions. By using NMR, we showed that only two of these interfaces exist in solution. By using FRET to characterize interactions on membranes, we showed that Syt1-SNARE complex binding primarily occurs via one of the remaining interfaces (primary interface), and this interaction is almost abolished in the presence of calcium. Together, these results suggest a model where, a release of Syt1-SNARE complex interactions by calcium triggers membrane fusion.Item A "garden variety" case of kidney stones viewed by a clinical physiologist(2015-09-25) Moe, Orson W.Item Management of postmenopausal osteoporosis(1982-10-14) Pak, Charles Y. C.Item Optical Quantal Analysis of Evoked and Spontaneous Single-Vesicle Fusion(2014-07-14) Leitz, Jeremy Thomas Sheng; Hilgemann, Donald W.; Hiesinger, Peter Robin; Albanesi, Joseph P.; Kavalali, Ege T.Synaptic vesicle recycling is critical for the maintenance and proper function of neurotransmission. Neurotransmission can proceed through action-potential evoked vesicle fusion where, upon depolarization, Ca2+ enters the nerve terminal though voltage-gated channels, interacts with vesicle-associated proteins to promote fusion with the terminal membrane, and causes release of vesicle contents. Neurotransmission can also occur spontaneously in the absence of stimulation, although this process is still Ca2+-dependent. Regardless of the mode of vesicle fusion, the vesicle lipids and protein components must be removed from the terminal membrane; the vesicle must be reconstituted and re-filled with neurotransmitter, so that it may ultimately be reused. Uncoupling the roles of Ca2+ in synaptic vesicle fusion and retrieval has been difficult to date as studies have relied on measurements of bulk synaptic vesicle retrieval. Here, to dissect the role of Ca2+ in these processes, we utilized low signal-to-noise pHluorin-tagged vesicular probes to monitor single synaptic vesicle recycling of both action-potential evoked and spontaneous fusion vesicles in rat hippocampal neurons. We show that during stimulation, increasing extracellular Ca2+ increases synaptic vesicle fusion probability, but decreases the rate of synaptic vesicle retrieval. This negative regulation of synaptic vesicle retrieval is blocked by the Ca2+ chelation as well as inhibition of calcineurin, a Ca2+-calmodulin-dependent phosphatase. Indeed, the slow time course of aggregate synaptic vesicle retrieval detected during repetitive activity can be explained by a progressive decrease in the rate of synaptic vesicle retrieval during the stimulation train. These results indicate Ca2+ entry during single action potentials slows the pace of subsequent synaptic vesicle recycling. Conversely, we found that synaptic vesicles that undergo spontaneous fusion are retrieved very rapidly and this retrieval time is Ca2+-independent. Interestingly, we found that within a single synaptic bouton, the rate of spontaneous neurotransmission is independent of evoked fusion probability, suggesting there are fundamental regulatory differences between these forms of neurotransmission. Moreover, we found that the glycoprotein Reelin can act presynaptically to enhance spontaneous neurotransmission without affecting evoked neurotransmission by mobilizing a molecularly specific subset of synaptic vesicles. These data illustrate fundamental differences in vesicle recycling between modes of neurotransmission at the single-vesicle level.Item Primary hyperparathyroidism and hypercalciuria: etiology and management(1973-09-20) Pak, Charles Y. C.Item Reciprocal Interactions of STIM1 with Orai1 and L-Type Ca2+ Channels in Cardiac Myocytes(2015-01-26) Nguyen, Derek; Luo, Xiang; Morales, Cyndi; Li, Dan; Gillette, Thoams; Hill, JosephBACKGROUND: Pathological cardiac hypertrophy can be triggered by abnormal Ca2+ levels. It has been shown that the mechanisms governing context-dependent changes in Ca2+ influx are linked to stromal interaction molecule 1 (STIM1). STIM1 is a sarcoplasmic reticulum Ca2+ sensor that regulates Ca2+ influx by directly activating store operated calcium channels such as Orai1 in response to stress such as intracellular Ca2+ depletion. STIM1 is also known to regulate L-type Ca2+ channels in cardiomyocytes, though the mechanism has not been elucidated. HYPOTHESIS: The Ca2+-sensing protein, STIM1, molecularly interacts with Orai1 and L-type Ca2+ channels in cardiomyocytes to facilitate Ca2+ influx as a master regulator of Ca2+ channels. METHODS: To address this, we monitored STIM1 interactions with either Orai1 or L-type Ca2+ channels in neonatal rat ventricular cardiomyocytes (NRVMs) and adult rat ventricular cardiomyocytes (ARVMs) using the Duolink in situ fluorescence assay. This technique utilizes proximity ligation assay technology to directly monitor interactions between proteins within 40nm. These channel interactions were observed under endogenous culture conditions as well as thapsigargin-mediated calcium store depleted conditions. Differences in STIM1 interactions between control (untreated) and thapsigargin-treated NRVM and ARVM cells were observed. RESULTS: In NRVM cells, STIM1 interactions with Orai1 increased by 44.79 ± 2.68% when treated with thapsigargin as compared to the control population (n=73-83). Conversely, STIM1 interactions with L-type Ca2+ channels decreased by 52.31 ± 3.45% compared to the control (n=64-69). In ARVM cells, STIM1 interactions with Orai1 decreased by 59.19 ± 2.39% when treated with thapsigargin as compared to the control population (n=11-16). STIM1 interactions with L-type Ca2+ channels on the other hand increased by 74.13 ± 0.21% compared to the control (n=11-19). CONCLUSIONS: We observed an opposite trend in STIM1 interactions with Orai1 and L-type Ca2+ channels in NRVM and ARVM cells. When treated with thapsigargin, their degree of interaction changed significantly. This would suggest that STIM1 undergoes dynamic changes in response to calcium conditions in order to regulate Ca2+ influx. At a molecular level, it would seem that STIM1 is a versatile Ca2+ channel regulator as it can alternately interact with both channels. Further studies will aid in the development of novel therapeutic strategies for the treatment of cardiac hypertrophy and disease.Item [Southwestern News](2003-07-17) Shields, AmyItem [Southwestern News](2002-04-11) Carter, WayneItem [Southwestern News](2004-12-28) Hansard, Donna StephItem [Southwestern News](1993-10-20) Gentry, LynnItem Spontaneous Neurotransmission as a Driver of Calcium Signaling(2016-09-20) Reese, Austin Lowell; Kim, Tae-Kyung; Kavalali, Ege T.; Monteggia, Lisa; Bezprozvanny, IlyaSpontaneous neurotransmission is a stochastic mode of neurotransmitter release that is not coupled to action potential arrival at the presynapse. Recent evidence has shed light upon the presynaptic and vesicular fusion machinery that makes this mode of release unique to action potential driven, or evoked release, yet the postsynaptic reception of these differing signals has yet to be probed in detail. Here, I illustrate that evoked and spontaneous release modes coexist within the same synapses, yet the neurotransmitter released falls upon separate subsets of postsynaptic receptors. As a consequence of this phenomenon, in glutamatergic hippocampal synapses the NMDA receptors activated by spontaneous release are able to drive large calcium transients that maintain synaptic homeostasis and suppress local protein synthesis.Item Standard Calcium Supplementation May Increase Kidney Stone Risk: A Study in Women with Postmenopausal Osteoporosis(2014-02-04) Menegaz, Colleen; Adams-Huet, Beverley; Li, Xilong; Rubin, CraigINTRODUCTION: The US prevalence of kidney stones has increased from 2.6% in 1972 to 8.4% in 2010.The majority of stones contain calcium (Ca) with hypercalciuria (hCa) highly associated with stone formation. Postmenopausal osteoporosis (pmo) is a common problem affecting 30% of postmenopausal women in the US. Supplementation with Ca and vitamin D (D) is recommended for most older women. PURPOSE: To determine the percentage of patients (pts) who become hypercalciuric while receiving Ca and D supplementation for pmo and to identify biochemical predictors for higher risk of developing hCa. METHODS: 84 ambulatory women aged ≥55 yrs in a RT comparing alendronate (ALN) and sustained-release sodium fluoride (SRF) for the tx of pmo received standard Ca and D supplements. 24-hr urinary Ca (uCa) and deoxypyridinoline and serum D, PTH and bone-specific alk. phos. were measured at 0 and 12 mos. We determined the percentage of pts who became hypercalciuric (uCa >250 mg/24h) during tx. to identify predictors of hCa. Changes in biochemical variables were assessed with mixed model repeated measures analysis. Logistic regression analysis was used to assess predictors of elevated uCa and construct receiver operating characteristic (ROC) curves. RESULTS: 42 pts were randomized to ALN and 42 to SRF. 67 pts completed ≥ one yr. 90% (27/30) of the ALN group and 92% (33/36) of the SRF group had normal uCa excretion at baseline. Patients with normal uCa at baseline experienced significant increases in uCa in the first year (ALN p=0.01, SRF p<0.0001). However, baseline hypercalciurics experienced no significant increase in uCa from baseline after Ca and D supplementation. In all, 13% (4/30) of ALN pts became hypercalciuric (p= 0.41) vs. 28% (10/36) in the SRF group (p=0.002). The best-fit multi-variable model determined baseline uCa (p=0.02) and D (p=0.03) were strong predictors of hCa at 12 mo. and produced a favorable ROC curve (0.90). Baseline uCa was a consistently strong predictor of hCa and a simple logistic regression analysis generated a ROC curve (0.84) which determined that 180 mg/d uCa at baseline was a strong predictive cut-point for detection of pts at higher risk of hCa with treatment. CONCLUSION: 21% of patients became hypercalciuric on recommended doses of Ca and D. Current Ca and D supplementation practice may have significant public health consequences by contributing to the growing incidence of nephrolithiasis. Practice guidelines should consider assessing baseline 24-hr uCa in all pts and 12 mo. 24-hr uCa in pts with baseline uCa ≥of 180 mg/24h.Item Studies on Plasma Membrane Proteins Involved in Membrane Traffic: Syntaxins and E-Syts(2007-05-22) Min, Sang-Won; Südhof, Thomas C.Fusion of synaptic vesicles is catalyzed by SNARE complex assembly which requires that the SNARE proteins syntaxin-1A and -1B, two isoforms of syntaxin-1, switch from a 'closed' to an 'open' conformation. To test the physiological significance of this switch, I analyzed mutant mice with a point mutation in syntaxin-1B which renders it predominantly 'open' in the syntaxin-1A null background. Whereas deletion of syntaxin-1A caused no detectable phenotype, opening of syntaxin-1B produced lethal epilepsy, independent of the presence of syntaxin-1A. Morphological and electrophysiological analyses revealed that opening of syntaxin-1B impaired steps in exocytosis upstream of vesicle priming, but enhanced Ca2+Item [UT News](1986-02-06) Bosler, Tommy JoyItem [UT Southwestern Medical Center News](2009-03-24) Shear, Kristen Holland