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Click to add/remove this article to your list of 'My Favorites' Reversible Translocation of Overexpressed STIM1-YFP is Coupled to Store-Operated Calcium Influx in Pancreatic beta-cells.

Year: 2007

Abstract Number: 0009-OR

Authors: NATALIA A. TAMARINA, ANDREY KUZNETSOV, LOUIS H. PHILIPSON, Chicago, IL

Results: Calcium (Ca2+) signaling regulates insulin secretion in pancreatic Ã[fnof]Ÿ-cells. STIM1, a transmembrane-spanning domain Ca2+-binding protein, has been proposed to function as an endoplasmic reticulum (ER) Ca2+ sensor regulating store-operated Ca2+ entry (SOCE). Here we demonstrate that in mouse insulinoma MIN6 cells, SOCE occurred in response to thapsigargin (Tg, 1 microM) and was blocked by 2-aminoethoxydiphenyl borate (2APB, 80% block at 100 microM). We hypothesed that the inhibition of SOCE by 2APB in MIN6 cells is mediated by its blocking of STIM-1 function. Sub-cellular distribution of overexpressed STIM1-YFP and its dependence on ER Ca2+ store depletion was studied by fluorescent confocal microscopy. Treatments that decrease ER Ca2+ concentration such as Tg (1 microM), cyclopiazonic acid (CPA, 50 microM), ionomycin (1 microM) in Ca2+ -free solution and carbachol (CCh, 250 microM) were associated with translocation of STIM1-YFP to a peri-plasma membrane (PM) location. To study the dynamics of STIM1-YFP near the PM, cells were photo-bleached using Total Internal Reflection Fluorescence (TIRF-FRAP) and the appearence of newly transported STIM1-YFP was recorded 5 minutes later. The appearance of STIM1-YFP at the plasma membrane was in highly defined areas of the plasma membrane, which were able to recover after photobleaching by insertion of new material. Treatment of cells with 2-APB (3.75-100 microM) before discharge of ER Ca2+ stores prevented the translocation of STIM1-YFP to the plasma membrane in a concentration-dependent and reversible manner, while application of 2-APB after STIM1-YFP translocation resulted in a restoration of its intracellular localization. We conclude that the translocation of Ca2+ -store depletion -regulated, STIM1-containing signaling complexes to their targets on the plasma membrane is a prerequisite of SOCE activation in MIN6 cells. The reversal of STIM1 translocation by 2APB may underlie the ability of this compound to interfere with critical aspects of Ca2+ signaling.