Immunosuppressant FK506 Research Articles

Targeting FKBP isoforms with small-molecule ligands

The FK506 binding protein (FKBP) family of proteins provide an interesting series of drug targets since different isoforms modulate diverse cellular pathways. There are therapeutic opportunities in the fields of cancer therapy, neurodegenerative conditions and psychiatric disorders. X-ray crystallographic or NMR data are available for eight of fourteen human FKBPs covering ten of the twenty-two different FKBP domains. We have made a detailed sequence and structural comparison of human FKBP domains. These data show that the chemical scaffolds common to the immunosuppressive inhibitors FK506 and rapamycin bind to the most conserved region of the binding site. This observation opens the way to the design of isoform specific inhibitors.

FKBP immunophilins and Alzheimer’s disease: A chaperoned affair

The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein-protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer's-disease-related pathways.

Transduced PEP-1-FK506BP Ameliorates Atopic Dermatitis in NC/Nga Mice

Immunophilin, FK506-binding protein 12 (FK506BP), is a receptor protein for the immunosuppressive drug FK506 by the FK506BP/FK506 complex. However, the precise function of FK506BP in inflammatory diseases remains unclear. Therefore, we examined the protective effects of FK506BP on atopic dermatitis (AD) in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced HaCaT cells and 2,4-dinitrofluorobenzene-induced AD-like dermatitis in Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice using a cell-permeable PEP-1-FK506BP. Transduced PEP-1-FK506BP significantly inhibited the expression of cytokines, as well as the activation of NF-κB and mitogen-activated protein kinase (MAPK) in TNF-α/IFN-γ-induced HaCaT cells. Furthermore, topical application of PEP-1-FK506BP to NC/Nga mice markedly inhibited AD-like dermatitis as determined by a histological examination and assessment of serum IgE levels, as well as cytokines and chemokines. These results indicate that PEP-1-FK506BP inhibits NF-κB and MAPK activation in cells and AD-like skin lesions by reducing the expression levels of cytokines and chemokines, thus suggesting that PEP-1-FK506BP may be a potential therapeutic agent for AD.

Effect of FK506 on the immunosuppression of mesenchymal stem cells induced by interferon-γ and tumor necrosis factor-α

Objective To investigate the mechanism of immunosuppressant FK506 on the immunesuppression of mesenchymal stem cells ( MSCs) induced by interferon ( IFN) -γ and tumor necrosis factor (TNF)-α. Methods MSCs pretreated with inflammatory cytokines were co-cultured with mice spleen cells. After incubation with FK506 for 72 h, proliferation of spleen cells was measured by methyl thiazol tetrazolium (MTT) assay and the apoptosis was assessed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to detect the major molecule that mediated the immunosuppression of MSCs and the effect of FK506 on the molecule. Results MSCs pretreated with inflammatory cytokines could inhibit the proliferation of spleen cells and increase the apoptosis of spleen cells, and the apoptosis rate was (53. 5 ±2. 5) % , which was higher than that of MSCs pretreated with IFN-γ or TNF-α and control group ( P ＜0. 05), and IFN-γ and TNF-α upregulated inducible nitric oxide synthase (iNOS) expression in MSCs significantly, and this immunosuppression could also be antagonized by a specific inhibitor 1400W. However,FK506 could inhibit iNOS expression in MSCs pretreated with IFN-γ and TNF-α, the inhibition of spleen cells proliferation by MSCs was reduced significantly, and the apoptosis rate of spleen cells was reduced to (44. 3 ±3. 2)% (P＜0. 05). Conclusion FK506 inhibits the immunosuppression of MSCs through downregulation of iNOS expression in MSCs pretreated with IFN-γ and TNF-α. Key words: Mesenchymal stem cells; Inflammation; Immunosuppression; FK506

FKBP38-Bcl-2 interaction: a novel link to chemoresistance

FKBP38, a noncanonical member of the immunosuppressive drug FK506 binding protein (FKBP) family members, possesses an inducible rotamase. FKBP38 interacts with several proteins and regulates multiple signaling pathways such as cell survival, apoptosis, proliferation, and metastasis. Deregulation of apoptosis is associated with chemoresistance and tumor relapse. The antiapoptotic protein Bcl-2 is a key player for increasing the apoptotic threshold in response to various cytotoxic drugs. The molecular interaction of Bcl-2 with FKBP38 potentiates the biological function of Bcl-2 and contributes to tumorigenesis and chemoresistance. Here, we discuss recent advances in the role of FKBP38 in connection with Bcl-2 and its possible link to chemotherapeutic resistance.

Mouse FKBP23 mediates conformer-specific functions of BiP by catalyzing Pro117cis/trans isomerization

FK506-binding proteins (FKBPs) are cellular receptors for the immunosuppressant FK506 and rapamycin. They belong to the ubiquitous peptidyl-prolyl cis/trans isomerases (PPIases) family, which can catalyze the cis/trans isomerization of peptidyl-prolyl bond in peptides and proteins. In previous work, we revealed that mouse FKBP23 binds immunoglobulin binding protein (BiP), the major heat shock protein (Hsp) 70 chaperone in the ER, and the binding is interrelated with [Ca2+]. Furthermore, the binding can suppress the ATPase activity of BiP through the PPIase activity of FKBP23. In this work, FKBP23 is demonstrated to mediate functions of BiP by catalyzing the Pro117cis/trans conformational interconversion in the ATPase domain of BiP. This result may provide new understanding to the novel role of PPIase as a molecular switch.

Targeting FK506 Binding Proteins to Fight Malarial and Bacterial Infections:Current Advances and Future Perspectives

There is an urgent need for the design and development of new and selective drugs for the treatment of malaria and bacterial infections as these pathogens are developing resistance to presently available therapies. Malaria is a life threatening disease in many countries and responsible for almost one million deaths annually. In particular, drug-resistant malarial parasites are hindering effective control of malaria and prompting to find novel druggable targets and develop compounds with mechanism of action different from the conventional drugs. In this quest, efforts were made to determine three-dimensional structures of Plasmodium falciparum and Plasmodium vivax FK506 binding proteins which bind the macrolides (FK506 and rapamycin) and also demonstrate peptidylprolyl cis-trans isomerase activity in a similar manner as human FKBP12. Previous studies revealed that the immunosuppressive drug FK506 exhibits potential anti-malarial activity by binding FK506 binding domains (FKBD). This review focuses on three different types of FK506 binding proteins/domains in pathogens, their structural characteristics and biological roles. Binding ability of these proteins with the macrolides has opened new possibilities to develop selective inhibitors for these novel targets to combat the life threatening infections.

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

Role of the Small GTPase Rho3 in Golgi/Endosome Trafficking through Functional Interaction with Adaptin in Fission Yeast

BackgroundWe had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast.Methodology/Principal FindingsIn the present study, we isolated rho3+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl− sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl−, and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner.Conclusions/SignificanceTaken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link between the small GTPase Rho and the clathrin-associated adaptor protein-1 in membrane trafficking.

Transduced PEP-1-FK506BP inhibits the inflammatory response in the Raw 264.7 cell and mouse models

FK506 binding protein 12 (FK506BP) is an immunophilin that acts as a receptor for the immunosuppressant drug FK506. Although the precise action of FK506BP remains unclear, it has emerged as a potential drug target for several inflammatory diseases. This study investigated the protective effects of FK506BP on inflammation in vitro and in vivo using protein transduction. A cell-permeable expression vector PEP-1-FK506BP was constructed. Lipopolysaccharide (LPS)- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated Raw 264.7 cells and ICR mice were treated with PEP-1-FK506BP. The expression of inflammatory response enzymes and cytokines was analyzed by Western blot, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and electrophoretic mobility shift assay. PEP-1-FK506BP efficiently transduced into Raw 264.7 cells and markedly inhibited the expression levels of cyclooxygenase-2 as well as pro-inflammatory cytokines. Furthermore, transduced PEP-1-FK506BP significantly reduced activation of nuclear factor-kappa B (NF-κB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the cells, whereas PEP-1-FK506BP reduced phosphorylation of p38 and extracellular signal-regulated kinase (ERK) in the animal models. These results indicate that PEP-1-FK506BP inhibits inflammatory response cytokines and enzymes by blocking NF-κB and MAPK including the phosphorylation of p38 and/or ERK MAPK in vitro and in vivo, suggesting that PEP-1-FK506BP may be a therapeutic agent against inflammatory skin diseases.

Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues.

The allyl moiety of the immunosuppressive agent FK506 is structurally unique among polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical, and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multistep enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogues, 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK506 analogues.

Overexpression of phospholipase Cε in keratinocytes upregulates cytokine expression and causes dermatitis with acanthosis and T‐cell infiltration

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases. We showed previously using PLCε-deficient mice that PLCε plays a critical role in activation of cytokine production in non-immune skin cells in a variety of inflammatory reactions. For further investigation of its role in inflammation, we created transgenic mice overexpressing PLCε in epidermal keratinocytes. The resulting transgenic mice spontaneously developed skin inflammation as characterized by formation of adherent silvery scales, excessive growth of keratinocytes, and aberrant infiltration of immune cells such as T cells and DC. Development of the skin symptoms correlated well with increased expression of factors implicated in human inflammatory skin diseases, such as IL-23, in keratinocytes, and with the accumulation of CD4(+) T cells producing IL-22, a potent inducer of keratinocyte proliferation. Intradermal injection of a blocking antibody against IL-23 as well as treatment with the immunosuppressant FK506 reversed these skin phenotypes, which was accompanied by suppression of the IL-22-producing T-cell infiltration. These results reveal a crucial role of PLCε in the development of skin inflammation and suggest a mechanism in which PLCε induces the production of cytokines including IL-23 from keratinocytes, leading to the activation of IL-22-producing T cells.

Pathogenic virus-specific T cells cause disease during treatment with the calcineurin inhibitor FK506: implications for transplantation.

Recently, several cases of fatal lymphocytic choriomeningitis virus (LCMV) infection occurred in transplant recipients being treated with the immunosuppressive calcineurin inhibitor FK506. These findings were surprising because LCMV is a noncytolytic virus. To understand how a noncytolytic virus can cause disease under conditions of immunosuppression, we used the mouse LCMV model and found that, similar to the observations in human transplant recipients, LCMV infection of FK506-treated mice resulted in a lethal disease characterized by viremia, lack of seroconversion, and minimal lymphocytic infiltrates in the tissues. However, despite the apparent absence of an antiviral immune response, this disease was orchestrated by virus-specific T cells. FK506 did not prevent the generation and proliferation of LCMV-specific T cells but instead altered their differentiation so that these effector T cells lost the ability to control virus but were still capable of mediating disease. These pathogenic T cells initiated a cytokine storm characterized by high levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6), and depletion of T cells or blockade of these inflammatory cytokines prevented the lethal disease. Our study shows that inhibiting calcineurin can generate pathogenic T cells and indicates that T cell-mediated viral disease can occur even under conditions of immunosuppression. Furthermore, we identify a potential strategy (blockade of TNF and IL-6) for treatment of transplant recipients who have acute complications of viral infection.

Subcellular rearrangement of hsp90‐binding immunophilins accompanies neuronal differentiation and neurite outgrowth

FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide-protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormone-dependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52•hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of βIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation.

ChemInform Abstract: Isolation of an Isomer of FK-506 from Fermentation of Streptomyces tsukubaensis and Its Chemical Synthesis from FK-506.

Abstract From the fermentation broth of Streptomyces tsukubaensis 9993 an isomer 2a of the immunosuppressant FK-506 1a was isolated as a minor metabolite. From NMR- and FAB-MS data follows that in 2a the size of the macrocycle is reduced by two carbon atoms due to a shift of the lactone-acyl from 0–26 to 0–24. The compound was synthesized from FK-506 and proved to be identical with the natural product 2a.

NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

The emergence of drug-resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X-ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.

Effects of immunosuppressant FK-506 on tooth movement

To test the hypothesis that immunosuppressant tacrolimus treatment can interfere with bone turnover and rate of tooth movement. One-hundred twenty Wistar male rats were divided into four groups: Group 1 (rats subjected to orthodontic movement plus treatment with saline solution vehicle), Group 2 (rats subjected to orthodontic movement plus treatment with FK506), Group 3 (rats treated with FK506 only), and Group 4 (rats treated with saline solution vehicle). The maxillary incisors were laterally moved with a reciprocal load of 35 cN. The dosage of FK506 was 2 mg/kg/day. Howship's lacunae, osteoclasts, and macrophages were counted. Tooth movement was found to be greater in Group 1 than in Group 2 for all time periods (on days 3, 7, and 14), although a significant difference was observed only on days 7 and 14 (p < 0.05). The number of osteoclasts was smaller in Group 1 than in Group 2, whereas the number of Howship's lacunae was greater. FK506 has the capacity of promoting osteoclasts inhibition with probable osteoclastic apoptosis of alveolar bone following tooth movement.

ChemInform Abstract: A Practical Synthesis of the Cyclohexyl Part of the Immunosuppressant FK506.

Starting from the benzylidene lactone 3 of D-(−)-quinic acid the cyclohexyl fragment 15 (C-28-C-34 part) of the immunosuppressant FK506 was synthesized. Key steps include homolytic deoxygenation reactions on compounds 4 and 6 as well as a regioselective opening of the benzylidene acetal 5. Opening of the lactone 7 to provide the methyl ester 8 was followed by methylation of the hydroxy group to give 9. Further steps provided the aldehyde 12 which was elongated to the alkyne 15. This sequence provides 15 in gram quantities.

Influência do imunossupressor tacrolimus (FK-506) na resistência à flexão de fêmures: estudo em ratos

OBJETIVO: Avaliar a resistencia a fratura de femures de ratos tratados com o imunossupressor tacrolimus FK-506 e compara-los a ratos tratados com placebo e nao tratados. METODOS: Foram utilizados 90 ratos da linhagem Wistar machos. Os animais tinham nove semanas de idade e peso entre 220 e 280g. O medicamento utilizado neste estudo foi o agente imunossupressor tacrolimus administrado por via oral, 2mg/kg/dia. A suspensao foi administrada com auxilio de seringa de insulina, sendo a dose terapeutica de manutencao suficiente para manter a atividade imunossupressora. Os animais foram divididos aleatoriamente em tres grupos (n = 30): grupo 1, nenhuma substância administrada; grupo 2, administracao do imunossupressor tacrolimus FK-506; e grupo 3, administrado apenas veiculo. O tratamento com FK-506 foi realizado por 28 dias. Foram avaliadas as contagens totais de leucocitos e diferencial (linfocitos, monocitos, eosinofilos e neutrofilos), com objetivo de monitoramento do efeito imunossupressor. Uma outra analise realizada foi a da densitometria ossea por absorcao de raios-X de dupla energia (DXA) antes e apos administracao do farmaco. Para avaliar a resistencia a flexao, desenvolveu-se dispositivo que serviu de apoio para realizacao dos ensaios mecânicos em maquina universal de ensaios mecânicos EMIC. RESULTADOS: Os resultados da resistencia a flexao demonstraram diferencas estatisticas entre os grupo 1 e 2 (p = 0,001) e 2 e 3 (p = 0,001). Nao foram encontradas diferencas estatisticas entre os grupos 1 e 3 (p = 0,995). CONCLUSOES: Os femures de ratos tratados com o imunossupressor apresentam menor resistencia mecânica que os de ratos normais e os que utilizaram placebo.

Store-depletion and hyperforin activate distinct types of Ca2+-conducting channels in cortical neurons

Cortical neurons embryos (E13) from murine brain have a wide diversity of plasma membrane Ca2+-conducting channels. For instance, they express several types of transient receptor potential channels of C-type (TRPC) and hyperforin, a potent TRPC6-channel activator, controls the activity of TRPC6-like channels. In addition, E13 cortical neurons possess plasma membrane channels activated in response to the depletion of internal Ca2+ pools. Since some TRPC channels seem to be involved in the activity of store-depletion-activated channels, we investigated whether hyperforin and the depletion of the Ca2+ stores control similar or distinct Ca2+ routes. Calcium imaging experiments performed with the fluorescent Ca2+ indicator Fluo-4 showed that the TRPC3 channel blocker Pyr3 potently inhibits with an IC50 of 0.5μM the entry of Ca2+ triggered in response to the thapsigargin-dependent depletion of the Ca2+ stores. On the other hand, Pyr3 does not block the hyperforin-sensitive Ca2+ entry. In contrast to the hyperforin responses, the Ca2+ entry through the store-depletion-activated channels is down-regulated by the competitive tyrosine kinase inhibitors genistein and PP2. In addition, the immunosuppressant FK506, known to modulate several classes of Ca2+-conducting channels, strongly attenuates the entry of Ca2+ through the store-depletion-activated channels, leaving the hyperforin-sensitive responses unaffected. Hence, the Zn2+ chelator TPEN markedly attenuated the hyperforin-sensitive responses without modifying the thapsigargin-dependent Ca2+ signals. Pyr3-insensitive channels are key components of the hyperforin-sensitive channels, whereas the thapsigargin-dependent depletion of the Ca2+ stores of the endoplasmic reticulum activates Pyr3-sensitive channels. Altogether, these data support the notion that hyperforin and the depletion of the Ca2+ pools control distinct plasma membrane Ca2+-conducting channels. This report further illustrates that, at the beginning of the corticogenesis, immature cortical neurons express diverse functional Ca2+ channels.