BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival.

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.

IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2.

Disrupting inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP3R-derived peptide (TAT-IDP(S))) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca(2+) signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP3R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP(S) in a more heterogeneous Bcl-2-dependent cancer model using a set of 'primed to death' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP(S) with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP(S) to promote IP3R-mediated Ca(2+) release. Although total IP3R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP3R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP(S)-induced Ca(2+) rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP3R2-protein level, but not with IP3R1-, IP3R3- or total IP3R-expression levels. Inhibiting or knocking down IP3R2 activity in SU-DHL-4-reduced TAT-IDP(S)-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP3R2 and to promote IP3-induced pro-apoptotic Ca(2+) signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP3R hyperactivity. In particular, cancer cells expressing high levels of IP3R2 are addicted to IP3R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca(2+) signaling and cell death.

Polycystins and cellular Ca2+ signaling.

The cystic phenotype in autosomal dominant polycystic kidney disease is characterized by a profound dysfunction of many cellular signaling patterns, ultimately leading to an increase in both cell proliferation and apoptotic cell death. Disturbance of normal cellular Ca(2+) signaling seems to be a primary event and is clearly involved in many pathways that may lead to both types of cellular responses. In this review, we summarize the current knowledge about the molecular and functional interactions between polycystins and multiple components of the cellular Ca(2+)-signaling machinery. In addition, we discuss the relevant downstream responses of the changed Ca(2+) signaling that ultimately lead to increased proliferation and increased apoptosis as observed in many cystic cell types.

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release.

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl.

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP(3)R) via its BH4 domain, thereby suppressing IP(3)R Ca(2+)-flux properties and protecting against Ca(2+)-dependent apoptosis. Here, we directly compared IP(3)R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP(3)R nor inhibited IP(3)-induced Ca(2+) release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP(3)R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP(3)R binding and inhibition. This difference in IP(3)R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP(3)Rs. In agreement with the IP(3)R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP(3)R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP(3)Rs and Ca(2+)-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca(2+) signaling and apoptosis.

Differential contribution of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.

Plasma membrane chloride (Cl(-)) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl(-) uptake mechanism) in Cl(-) handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl(-) through a bumetanide- and Na(+)-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl(-) during GABA(A) receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl(-) concentration (i.e., [Cl(-)](i) decrease) was similar in both cell types and was unaffected by lowering the extracellular Na(+) concentration. In contrast, the recovery on depletion (i.e., [Cl(-)](i) increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na(+). The experimental observations could be reproduced by a mathematical model for intracellular Cl(-) kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl(-)-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl(-)](i), whereas lower NKCC1 activity in MNs allows large [Cl(-)](i) variations during neuronal activity.

Pathophysiology of nasal obstruction and meta-analysis of early and late effects of levocetirizine.

Nasal obstruction, also referred to as congestion, blockage or stuffiness, is a crucial symptom in allergic rhinitis (AR) and may affect sleep as well as quality of life. Early- and late-phase-allergic reactions both contribute to nasal obstruction, although it primarily represents a major symptom in the chronic allergic reaction. A complex network of inflammatory and neurogenic phenomena relates to chronic nasal obstruction, including the subepithelial accumulation of inflammatory cells, particularly mast cells and eosinophils, and the release of neuropeptides. Nasal obstruction is a difficult-to-treat symptom. Vasoconstrictors (decongestants) and intranasal corticosteroids, due to their anti-inflammatory properties, have mainly been used for relieving the nasal passages from the congested mucosa. However, there is accumulating evidence recently that the latest-generation potent antihistamines have decongestant properties in AR. This paper aims to review the pathophysiologic background of nasal obstruction and the evidence for an antihistamine, levocetirizine, in relieving nasal congestion. A meta-analysis on the early and late effects of levocetirizine on nasal obstruction under artificial and natural allergen exposure conditions is presented, demonstrating convincingly that levocetirizine shows a consistent effect on nasal obstruction as early as over the first 2 h and sustained over 6 weeks.

Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells.

The Golgi apparatus is, like the endoplasmic reticulum, an inositol-1,4,5-trisphosphate-sensitive Ca2+ store, but its role in setting up Ca2+ signals is not well understood. We have now measured histamine-induced Ca2+ signals in HeLa cells pretreated with brefeldin A, a fungal metabolite that leads to the fragmentation and subsequent disappearance of the Golgi apparatus by its reabsorption within the endoplasmic reticulum. Ca2+ responses in which the free cytoplasmic Ca2+ concentration returned to resting levels during the histamine stimulation (mainly baseline Ca2+ oscillations or a single Ca2+ peak) occurred more often in brefeldin A pretreated cells, resulting in a lower Ca2+ plateau in population measurements. The latencies before the onset of the Ca2+ signals were longer after brefeldin A pretreatment. These results suggest that the integrity of the Golgi apparatus contributes to the shaping of intracellular Ca2+ signals.

[Intracellular calcium-releasing channels as cellular targets for immunophilins: a molecular, functional and structural analysis]. / Intracellulaire calcium-vrijzettingskanalen als cellulaire doelwitten voor immunofilines: een moleculaire, functionele en structurele analyse.

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.

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments.

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.

Inositol trisphosphate producing agonists do not mobilize the thapsigargin-insensitive part of the endoplasmic-reticulum and Golgi Ca2+ store.

Non-mitochondrial intracellular Ca2+ stores contain both thapsigargin-sensitive sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and thapsigargin-insensitive secretory-pathway Ca2+-ATPases (SPCA1). We now have studied the Ca2+-release properties of the compartments associated with these pumps in intact, i.e. non-permeabilized, cells of different origin (HeLa, keratinocytes, 16HBE14o-, COS-1, A7r5) and with different approaches (45Ca2+ fluxes, Ca2+ imaging and measurements of the free luminal [Ca2+] in the endoplasmic-reticulum and the Golgi apparatus using targeted aequorin). Application of an extracellular agonist in the absence of thapsigargin induced in all cells a Ca2+ release from both the endoplasmic-reticulum and the Golgi apparatus. The agonists were not able to release Ca2+ in the presence of 10 microM thapsigargin, except in COS-1 cells overexpressing SPCA1, where this pump not only appeared in the Golgi compartment but also overflowed into the agonist-sensitive part of the endoplasmic-reticulum. We conclude that the subcompartments of the endoplasmic-reticulum and of the Golgi complex that endogenously express SPCA1 are insensitive to agonist stimulation.

Calcineurin and intracellular Ca2+-release channels: regulation or association?

The Ca(2+)- and calmodulin-dependent phosphatase calcineurin was reported to interact with the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor (RyR) and to modulate their phosphorylation status and activity. However, controversial data on the molecular mechanisms involved and on the functional relevance of calcineurin for these channel-complexes have been described. Hence, we will focus on the functional importance of calcineurin for IP(3)R and RyR function and on the different mechanisms by which Ca(2+)-dependent dephosphorylation can affect the gating of those intracellular Ca(2+)-release channels. Since many studies made use of immunosuppressive drugs that are inhibiting calcineurin activity, we will also have to take the different side effects of these drugs into account for the proper interpretation of the effects of calcineurin on intracellular Ca(2+)-release channels. In addition, it became recently known that various other phosphatases and kinases can associate with these channels, thereby forming macromolecular complexes. The relevance of these enzymes for IP(3)R and RyR functioning will be reviewed since in some cases they could interfere with the effects ascribed to calcineurin. Finally, we will discuss the downstream effects of calcineurin on the regulation of the expression levels of intracellular Ca(2+)-release channels as well as the relation between IP(3)R- and RyR-mediated Ca(2+) release and calcineurin-dependent gene expression.

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1.

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.

Pharmacology of inositol trisphosphate receptors.

In almost all cells, cytosolic Ca(2+) is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca(2+) release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP(3)), which interacts with the IP(3) receptor (IP(3)R), a tetrameric intracellular Ca(2+)-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP(3)R, including the IP(3)-binding domain, calmodulin-binding sites, adenine nucleotide-binding sites and the sites for interaction for FK506-binding proteins and other regulators. We will particularly focus on the pharmacological tools that interfere with these domains and discuss their relative specificity for the IP(3)R, thereby indicating their potential usefulness for unraveling the complex functional regulation of the IP(3)R.

IP(3)-mediated Ca(2+) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor.

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.

Washing out of lipophilic compounds induces a transient increase in the passive Ca(2+) leak in permeabilized A7r5 cells.

We have investigated how the immunosuppressant drug FK506 affected the basal Ca(2+) leak in permeabilized A7r5 cells. Non-mitochondrial Ca(2+) stores loaded to steady state with Ca(2+) slowly lost their accumulated Ca(2+) during incubation in a Ca(2+)-free efflux medium. FK506 up to 100 microM had no effect on the basal Ca(2+) leak. In contrast, the rate of Ca(2+) release proceeded much faster immediately after washing out FK506. The increase in rate of Ca(2+) release after washing out of this compound depended on both its initial concentration and on the time of pre-incubation. A similar effect was also observed after removing another immunosuppressant drug (rapamycin) and after removing the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin C. Since all these substances have a high octanol/H(2)O partition coefficient and accumulate in the endoplasmic reticulum membrane, we suggest that the transient increase in the basal Ca(2+) leak is due to the sudden removal of these lipophilic substances from the membrane.

Calcium puffs are generic InsP(3)-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses.

Elementary Ca(2+) signals, such as "Ca(2+) puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca(2+) signalling. We characterized Ca(2+) puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca(2+) puffs in these cell types suggests that they are a generic elementary Ca(2+) signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca(2+) signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca(2+) puffs. The amplitude and duration of Ca(2+) puffs were substantially reduced. In addition, the number of Ca(2+) puff sites active during the onset of a Ca(2+) wave declined. The consequence of the changes in Ca(2+) puff properties was that cells displayed a lower propensity to trigger regenerative Ca(2+) waves. Therefore, Ca(2+) puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.

The conserved sites for the FK506-binding proteins in ryanodine receptors and inositol 1,4,5-trisphosphate receptors are structurally and functionally different.

We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP(3)R1 and IP(3)R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind approximately 30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg(2+), and weakened in the absence of Ca(2+) but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val(2322), located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val(2322) for leucine (as in IP(3)R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val(2322) for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as alpha-helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP(3)R isoforms. A chimeric RyR3/IP(3)R1, containing the core of the FKBP12-binding site of IP(3)R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP(3)Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca(2+)-release channels. Structural differences in the FKBP-binding site of RyRs and IP(3)Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP(3)Rs.