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STIM1 and Orai1 are essential components of Ca(2+) release-activated Ca(2+) channels (CRACs). After endoplasmic reticulum Ca(2+) store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and(More)
Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a(More)
Pancreatic beta-cells maintain glucose homeostasis by their regulated Ca(2+)-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca(2+) levels, but their relative significance in mouse beta-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca(2+) ([Ca(2+)](i)) clearance after brief,(More)
STIM1 and Orai1 have recently been identified to be crucial in the regulation of store-operated Ca(2+) entry. However, it remains to be established how STIM1 couples store depletion to the functioning of Orai1 in the plasma membrane. Using quantitative measurement, we find little STIM1 on the surface membrane which is not increased by store depletion. We(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 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)
Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway.(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 cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization.(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)