Abstract
Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.
Highlights
Store-operated calcium entry (SOCE),4 a Ca2ϩ influx mechanism regulated by the filling state of the intracellular Ca2ϩ stores, is a major mechanism for Ca2ϩ influx in non-excitable cells (1)
Our previous studies have reported that depletion of the acidic Ca2ϩ stores using TBHQ results in the activation of a SOCE pathway that is regulated differently from SOCE controlled by the dense tubular system (DTS) (32)
The expression of STIM1 in acidic organelles is supported by co-immunoprecipitation of this protein with the SERCA3 isoform, which has been reported to be located in the acidic stores on the basis of its sensitivity to TBHQ (42) and the sensitivity of TBHQ-evoked Ca2ϩ release to bafilomycin A1 (4)
Summary
Materials—Fura-2 acetoxymethyl ester (fura-2/AM), Lysosensor Green DND-189, calcein/AM, and Alexa Fluor 350- and 647conjugated secondary antibodies were from Invitrogen. Fluorescence was recorded from 2-ml aliquots of magnetically stirred cellular suspension (2 ϫ 108 platelets/ml) at 37 °C using a Cary Eclipse spectrophotometer (Varian Ltd., Madrid, Spain) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. To detect the primary antibody, blots were incubated for 45 min with horseradish peroxidase-conjugated ovine anti-mouse IgG antibody or horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody diluted 1:10,000 in TBST and exposed to enhanced chemiluminiscence reagents for 5 min. Platelets were incubated with antibodies for an additional 60 min at 37 °C and centrifuged at 350 ϫ g for 20 min and resuspended in HBS prior to the experiments. Samples were incubated with rabbit anti-Orai and mouse anti-STIM1 antibodies overnight at room temperature in PBS containing 0.5% BSA as blocking agent, followed by incubation with Alexa Fluor 350- and 647conjugated secondary antibodies for 1 h. A one-way analysis of variance combined with the Tukey tests was used. p Ͻ 0.05 was considered to be significant for a difference
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