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The Ca(2+) release-activated Ca(2+) (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca(2+) store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain).(More)
Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH(More)
Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma(More)
The existence of clathrin-independent recycling of secretory vesicles has been controversial. By combining patch-clamp capacitance recording, optical methods and specific molecular interventions, we dissect two types of mechanistically different endocytosis in pancreatic beta cells, both of which require GTP and dynamin. The fast one is a novel(More)
Recent studies have identified STIM1 and Orai1 as essential and conserved components of the Ca2+ release-activated Ca2+ (CRAC) channel. However, the reason STIM1 exhibits different distributions in nematode Caenorhabditis elegans and in human cells before endoplasmic reticulum (ER) calcium store depletion has not been clarified. Compared to the diffuse ER(More)
Glucose supply fluctuates between meal and fasting periods and its consumption by the body varies greatly depending on bodily metabolism. Pancreatic islets of Langerhans secrete various endocrine hormones including insulin and glucagon to keep blood glucose level relatively constant. Additionally, islet hormones regulate activity of neighboring cells as(More)
Three different methods, membrane capacitance (C(m)) measurement, amperometry and FM dye labeling were used to investigate the role of extracellular ATP in insulin secretion from rat pancreatic beta cells. We found that extracellular application of ATP mobilized intracellular Ca(2+) stores and synchronously triggered vigorous exocytosis. No influence of ATP(More)
Pancreatic islet cells use neurotransmitters such as l-glutamate to regulate hormone secretion. We determined which cell types in mouse pancreatic islets express ionotropic glutamate receptor channels (iGluRs) and describe the detailed biophysical properties and physiological roles of these receptors. Currents through iGluRs and the resulting membrane(More)
Glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells is critical to the maintenance of blood glucose homeostasis in animals. Both decrease in pancreatic beta-cell mass and defects in beta-cell function contribute to the onset of diabetes, although the underlying mechanisms remain largely unknown. Molecular imaging techniques can help(More)
Recent advent of light-sheet fluorescent microscopy (LSFM) has revolutionized three-dimensional biological imaging with high temporal resolution and minimal photodamage, enabling long-term fluorescence imaging of tissues and small organisms [1-2]. By combining two-photon fluorescence excitation with LSFM, Truong et al. [3] have created a two-photon digital(More)