The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca 2؉ storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca 2؉ that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca 2؉ store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca 2؉ uptake. Direct measurements with ER-targeted Ca 2؉ -sensitive fluorescent ''cameleon'' proteins revealed that Bcl-2 decreased the free Ca 2؉ concentration within the lumen of the ER, [Ca 2؉ ]ER. Analysis of the kinetics of Ca 2؉ store depletion in response to the Ca 2؉ -ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca 2؉ concentration within the ER lumen by increasing the Ca 2؉ permeability of the ER membrane. The increased ER Ca 2؉ permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane. T he protooncogene Bcl-2 protects cells against apoptosis (1).Morphological and biochemical studies demonstrate that Bcl-2 has two major intracellular localizations (2-5): (i) mitochondria and (ii) the endoplasmic reticulum (ER). Mitochondria have received major attention as organelles involved in apoptosis. However, Bcl-2 not only prevents the mitochondrial permeability switch and the subsequent release of cytochrome c (6-8), but also inhibits apoptosis in response to microinjected cytochrome c (9, 10). This finding suggests that Bcl-2 inhibits apoptotic mechanisms downstream of cytochrome c, possibly at the level of the ER. Consistent with this hypothesis, a recent study demonstrated that ER-targeted Bcl-2 was able to inhibit apoptosis induced by Myc in a rat fibroblast cell line (11).The ] c ) (12, 13) and the Ca 2ϩ permeability of the plasma membrane (store-operated Ca 2ϩ influx) (14,15). The function of Bcl-2 at the ER is only poorly understood. However, several arguments point toward the possibility that Bcl-2 might alter ER ionic homeostasis: (i) the three-dimensional structure of Bcl-2-related proteins is reminiscent of pore-forming bacterial toxins (16,17), (ii) Bcl-2 can function as an ion channel in artificial lipid bilayers (18-21), and (iii) alteration of cellular Ca 2ϩ signaling by Bcl-2, compatible with altered ER Ca 2ϩ homeostasis, has been reported in several studies (22)(23)(24)(25).In this study, we have investigated the effect of Bcl-2 on (i) the regulation of [Ca 2ϩ -free medium contained no CaCl 2 , but 0.5 mM EGTA. When DMSO was used as a drug solvent, the final concentration in the recording medium did not exceed 0.1%.Bcl-2-Expressing Cell Lines. Mouse lymphoma A20 cells, stabl...

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Neutrophils kill microbes with reactive oxygen species generated by the NADPH oxidase, an enzyme which moves electrons across membranes. Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established. We show that neutrophils from VSOP/Hv1−/− mice lack proton currents but have normal electron currents, indicating that these cells have a fully functional oxidase that cannot conduct protons. VSOP/Hv1−/− neutrophils had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice. Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin. Loss of VSOP/Hv1 also aborted calcium responses to chemoattractants, increased neutrophil spreading, and decreased neutrophil migration. The migration defect was restored by the addition of a calcium ionophore. Our findings indicate that proton channels extrude the acid and compensate the charge generated by the oxidase, thereby sustaining calcium entry signals that control the adhesion and motility of neutrophils. Loss of proton channels thus aborts superoxide production and causes a severe signaling defect in neutrophils.

To study Ca 2؉ fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in ϳ20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe

A newly identified splice variant of STIM1 called STIM1L forms constitutive clusters that interact with actin and Orai1 and allows fast repetitive Ca2+ release.

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca 2؉ ] in involving storeoperated Ca 2؉ entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca 2؉ sensor activating Orai Ca 2؉ channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca 2؉ ] in and intracellular Ca 2؉ store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K ؉ channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

To study the role of calreticulin in Ca 2؉ homeostasis and apoptosis, we generated cells inducible for fulllength or truncated calreticulin and measured Ca 2؉ signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators.

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