We have characterised the functional regulation of the type-3 ryanodine receptor by the 12 kDa FK506-binding protein. Wild-type type-3 ryanodine receptor and mutant type-3 ryanodine receptor in which the critical valine at position 2322 in the central 12 kDa FK506-binding protein binding site was substituted by aspartate, were stably expressed in human embryonic kidney cells. In contrast to the wild-type receptor, the mutant receptor was strongly impaired in binding to immobilised glutathione S-transferase 12 kDa FK506-binding protein. Caffeine-induced 45Ca(2+)-efflux was markedly increased in cells expressing mutant type-3 ryanodine receptor whereas the maximal-releasable Ca2+ was not affected. Confocal Ca2+ imaging provided clear evidence for a much higher sensitivity of the mutant receptor, which showed global Ca2+ release at about 20-fold lower caffeine concentrations than the wild-type receptor. Spontaneous Ca2+ sparks were observed in both wild-type- and mutant-expressing cells but the number of sparking cells was about 1.5-fold higher in the mutant group, suggesting that the degree of FK506 binding controls the stability of the closed state of ryanodine receptor channels. Furthermore, overexpression of 12 kDa FK506-binding protein decreased the number of sparking cells in the wild-type-expressing cells whereas it did not affect the number of sparking cells in cells expressing the mutant receptor. Concerning spark properties, the amplitude and duration of Ca2+ sparks mediated by mutant channels were significantly reduced in comparison to wild-type channels. This suggests that functional coupling between different mutant type-3 ryanodine receptor channels in a cluster is impaired. Our findings show for the first time that the central binding site for the 12 kDa FK506-binding protein of type-3 ryanodine receptor, encompassing the critical valine proline motif, plays a crucial role in the modulation of the Ca2+ release properties of the type-3 ryanodine receptor channel, including the regulation of both global Ca2+ responses and spontaneous Ca2+ sparks.

In this study, the FKBP12-binding properties of IP3Rs and RyRs were compared. Although the primary sequence of IP3Rs en RyRs contained a putative FKBP12-binding site, the functional, molecular and structural properties of these sites appeared to be completely different. For RyRs, FKBPs appear to function as associated proteins that are important for the functional regulation of the channel, thereby stabilizing the RyR complex. For IP3Rs, FKBPs might be involved in the de novo protein synthesis of the IP3Rs and the folding of the peptide chain to a functional IP3R protein, thereby functioning as helper enzymes. Hence, it is very unlikely that they function as associated regulatory proteins of the IP3R. In addition, we provided evidence that FKBP 12 is also an important regulating protein of the Ca(2+)-flux properties of the RyR3. FKBP12 clearly modulated both RyR3-mediated global and local Ca(2+)-responses.

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.

In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were differentially localized. IP3R1 was predominantly localized in the perinuclear region, whereas IP3R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 μM argininevasopressin induced a redistribution of IP3R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP3R3 appeared to be unaffected. The redistribution process occurred independently of IP3R downregulation. No structural changes of the endoplasmic reticulum were observed, but SERCA-type Ca2+ pumps redistributed similarly to IP3R1. The change in IP3R1 localization induced by arginine-vasopressin could be blocked by the simultaneous addition of nocodazole or taxol and depended on Ca2+ release from intracellular stores since Ca2+-mobilizing agents such as thapsigargin and cyclopiazonic acid could induce the redistribution. Furthermore, various protein kinase C inhibitors could inhibit the redistribution of IP3R1, whereas the protein kinase C activator 1-oleoyl-2-acetyl-sn-glycerol induced the redistribution. Activation of protein kinase C also induced an outgrowth of the microtubules from the perinuclear region into the cytoplasm, similar to what was seen for the redistribution of IP3R1. Finally, blocking vesicular transport at the level of the intermediate compartment inhibited the redistribution. Taken together, these findings suggest a role for protein kinase C and microtubuli in the redistribution of IP3R1, which probably occurs via a mechanism of vesicular trafficking.

We studied the role of the Pmr1-containing Ca(2+) store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100 microM ATP with baseline Ca(2+) spiking, while non-transfected cells produced an initial Ca(2+) peak followed by a long-lasting plateau. The Ca(2+) signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca(2+) with a regenerative Ca(2+) signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca(2+) concentration. These results demonstrate the importance of the Pmr1-containing Ca(2+) store in generating or modifying cellular Ca(2+) signals.

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), were stably transfected with IP(3)R type 3 (IP(3)R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP(3)Rs in transfected and selected high expression cloned cells. Using confocal Ca(2+) imaging, carbachol (CCh)-induced Ca(2+) release signals were studied. Low CCh concentrations (< or = 750 nM) evoked baseline Ca(2+) oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca(2+) responses varied between fast, large Ca(2+) spikes and slow, small Ca(2+) humps, while in the clone only Ca(2+) humps were observed. Ca(2+) humps in the transfected cells were associated with a high expression level of IP(3)R3. At high CCh concentrations (10 microM) Ca(2+) transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP(3)R3 lacked the C-terminal channel domain but IP(3)-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP(3)Rs but some protein degradation was also observed. We conclude that in transfected cells expression of functional type 3 IP(3)Rs causes an apparent higher affinity for IP(3). In the clone, the presence of degraded receptors leads to an efficient cellular IP(3) buffer and attenuated IP(3)-evoked Ca(2+) release.

Cytosolic Ca2+ oscillations can be due to cycles of release and re-uptake of internally stored Ca2+. To investigate the nature of these Ca2+stores, we expressed the Pmr1 Ca2+ pump ofCaenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca2+ uptake by the sarco(endo)plasmic reticulum Ca2+-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca2+ store that was less leaky for Ca2+ than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca2+ with a regenerative Ca2+ signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca2+ spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca2+-ATPase. Abortive Ca2+ spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca2+ store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca2+ spiking.

Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP(3)-mediated Ca(2+) release signals in human SH-SY5Y neuroblastoma cells. [Ca(2+)](i) increases were not affected by ryanodine (30 microgM) or caffeine (10 mM) and largely insensitive to removal of external Ca(2+), indicating predominance of IP(3)-induced Ca(2+) release. Ca(2+) signals evoked by high concentration (10 microM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca(2+) changes in most cells displayed striking spatiotemporal heterogeneity. The Ca(2+) response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca(2+) changes in processes either occurred in a homogeneous manner throughout the whole process or were sometimes confined to hot spots. Regional differences in surface-to-volume ratio appear to be critical clues that determine the spatiotemporal pattern of intracellular Ca(2+) release signals.

It is still debated whether inositol 1,4,5-trisphosphate(IP3)-induced Ca2+ release is loading-dependent. We now report that stimulation of the IP3 receptor by luminal Ca2+ depends on the cytosolic [Ca2+] in permeabilized A7r5 cells. The EC50 and maximal extent of Ca2+ release were loading-dependent in the presence of 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid: the EC50 increased 1.9-fold and the maximal release decreased from 88 to 52% when the stores contained 73% less Ca2+. In the presence of 0.3 μM free Ca2+, the EC50 for filled and less filled stores differed, however, only 1.2-fold and the maximal Ca2+ release was respectively 96 and 87% of the total releasable Ca2+. At 1 μM free Ca2+, the difference in EC50 between filled and less filled stores again became larger (2.2-fold)and the maximal Ca2+ release decreased from 93 to 87% when the stores contained less Ca2+.