Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

In polarized epithelial cells [Ca 2؉ ] i waves are initiated in discrete regions and propagate through the cytosol. The structural basis for these compartmentalized and coordinated events are not well understood. In the present study we used a combination of [Ca 2؉ ] i imaging at high temporal resolution, recording of Ca 2؉ -activated Cl ؊ current, and immunolocalization by confocal microscopy to study the correlation between initiation and propagation of [Ca 2؉ ] i waves and localization of Ca 2؉ release channels in pancreatic acini and submandibular acinar and duct cells. In all cells Ca 2؉ waves are initiated in the luminal pole and propagate through the cell periphery to the basal pole. All three cell types express the three known inositol 1,4,5-trisphosphate receptors (IP 3 Rs). Expression of IP 3 Rs was confined to the area just underneath the luminal and lateral membranes, with no detectable receptors in the basal pole or other regions of the cells. In pancreatic acini and SMG ducts IP 3 R3 was also found in the nuclear envelope. Expression of ryanodine receptor was detected in submandibular salivary gland cells but not pancreatic acini. Accordingly, cyclic ADP ribose was very effective in mobilizing Ca 2؉ from internal stores of submandibular salivary gland but not pancreatic acinar cells. Measurement of [Ca 2؉ ] i and localization of IP 3 Rs in the same cells suggests that only a small part of IP 3 Rs participate in the initiation of the Ca 2؉ wave, whereas most receptors in the cell periphery probably facilitate the propagation of the Ca 2؉ wave. The combined results together with our previous studies on this subject lead us to conclude that the internal Ca 2؉ pool is highly compartmentalized and that compartmentalization is achieved in part by polarized expression of Ca 2؉ channels.

The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca 2؉ pumps and characterizing their role in initiation and propagation of Ca 2؉ waves. Specific and polarized expression of Ca 2؉ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca 2؉ release with ionomycin on Ca 2؉ release evoked by agonists and Ca 2؉ uptake induced by antagonists. Despite their polarized expression, Ca 2؉ uptake by SERCA pumps and Ca 2؉ efflux by PMCA resulted in uniform reduction in [Ca 2؉ ] i . Surprisingly, inhibition of the SERCA pumps, but not Ca 2؉ release by ionomycin, eliminated the distinct initiation sites and propagated Ca 2؉ waves, leading to a uniform increase in [Ca 2؉ ] i . In addition, inhibition of SERCA pumps reduced the rate of Ca 2؉ release from internal stores. The implication of these findings to rates of Ca 2؉ diffusion in the cytosol, compartmentalization of Ca 2؉ signaling complexes, and mechanism of Ca 2؉ wave propagation are discussed.The agonist-evoked Ca 2ϩ signal is governed by the coordinated action of Ca 2ϩ channels and Ca 2ϩ pumps (1). In recent years it has become clear that [Ca 2ϩ ] i changes occur in the form of elemental events emanating in specialized microdomains and propagating as Ca 2ϩ waves (see reviews in Ref. 2). Such behavior was also described in pancreatic acinar cells (3-6) in which the [Ca 2ϩ ] i wave is initiated in the luminal pole (LP) 1 and propagates to the basal pole. In the preceding manuscript (7) we showed similar phenomena in SMG acinar and duct cells. Characterization and localization of Ca 2ϩ release channels in the three cell types (7) suggests that the channels determine the initiation site and pattern of the [Ca 2ϩ ] i waves. Thus, the cells express all three types of IP 3 R and the SMG cells also express the ryanodine receptor. Expression of the receptors appears to be confined to the area just underneath the luminal and lateral membranes (7).The type of Ca 2ϩ pumps expressed in each cell type and their role in control of the [Ca 2ϩ ] i wave is not known. Functional studies showed the presence of PMCA in pancreatic acini (8, 9), which is activated by agonist stimulation (8). In several studies isolated membranes and vesicles were used to localize the PMCA in the basolateral membrane of these cells (10). However, a recent study in which external [Ca 2ϩ ] was measured suggested that Ca 2ϩ pumping across the luminal membrane dominat...

Members of the bcl-2 gene family encode proteins that function either to promote or to inhibit apoptosis. Despite numerous eorts, the mechanism of action of Bcl-2, an anti-apoptotic protein, is still not clear. In particular, the relation between Bcl-2 and the endoplasmic reticulum (ER) calcium store is not well-understood. In the present work, we examined the eect of Bcl-2 on the ER store. We demonstrate that overexpression of Bcl-2 in breast epithelial cells modulates ER store by upregulating calcium pump (SERCA) expression without aecting the release channel (IP 3 R). The steady state levels of SERCA2 mRNA and protein were both increased in Bcl-2 expression clones. The increase in SERCA2 protein leads to accelerated calcium uptake and enhanced Ca 2+ loading. In addition, we also show the detection of intracellular interaction between Bcl-2 and SERCA molecules by co-immunoprecipitation. Since high lumenal Ca 2+ concentration of ER is essential for normal cell functions, the results suggest that Bcl-2 preserves the ER Ca 2+ store by upregulating SERCA gene expression as well as by a possible interaction with the pump.

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