Functional properties of recombinant type I and type III inositol 1, 4,5-trisphosphate receptor isoforms expressed in COS-7 cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are ubiquitous intracellular Ca(2+) release channels whose functional characterization by transfection has proved difficult due to the background contribution of endogenous channels. In order to develop a functional assay to measure recombinant channels, we transiently transfected the rat type I IP(3)R into COS-7 cells. Saponin-permeabilized COS cells transfected with type I IP(3)R showed a 50% increase in inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release at saturating [IP(3)] (10 micrometer) but no enhancement at subsaturating [IP(3)] (300 nm). However, cotransfection of the IP(3)R and human sarco/endoplasmic reticulum ATPase (SERCA)-2b ATPase cDNA resulted in 60 and 110% increases in Ca(2+) release at subsaturating and saturating doses of IP(3), respectively. IP(3) or adenophostin A failed to release (45)Ca(2+) from microsomal vesicles prepared from cells expressing either type I IP(3)R or SERCA cDNAs alone. However, microsomal vesicles prepared from cells doubly transfected with IP(3)R and SERCA cDNAs released 33.0 +/- 0.04% of the A23187-sensitive pool within 30 s of 1 micrometer adenophostin A addition. Similarly, the initial rate of (45)Ca(2+) influx into oxalate-loaded microsomal vesicles was inhibited by IP(3) only when the microsomes were prepared from COS cells doubly transfected with SERCA-2b and IP(3)R DNA. The absence of a functional contribution from endogenous IP(3)Rs has enabled the use of this assay to measure the Ca(2+) sensitivities of IP(3)-mediated (45)Ca(2+) fluxes through recombinant neuronal type I (SII(+)), peripheral type I (SII(-)), and type III IP(3)Rs. All three channels displayed a biphasic dependence upon [Ca(2+)](cyt). Introduction of mutations D2550A and D2550N in the putative pore-forming region of the type I IP(3)R inhibited IP(3)-mediated (45)Ca(2+) fluxes, whereas the conservative substitution D2550E was without effect. This assay therefore provides a useful tool for studying the regulatory properties of individual IP(3)R isoforms as well as for screening pore mutations prior to more detailed electrophysiological analyses.     

Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt20 cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt20 cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2+ release from ER by IP3Rs within cells.     

Akt kinase phosphorylation of inositol 1,4,5-trisphosphate receptors

A consensus RXRXX(S/T) substrate motif for Akt kinase is conserved in the C-terminal tail of all three inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) isoforms. We have shown that IP3R can be phosphorylated by Akt kinase in vitro and in vivo. Endogenous IP3Rs in Chinese hamster ovary T-cells were phosphorylated in response to Akt activation by insulin. LnCAP cells, a prostate cancer cell line with constitutively active Akt kinase, also showed a constitutive phosphorylation of endogenous type I IP3Rs. In all cases, the IP3R phosphorylation was diminished by the addition of LY294002, an inhibitor of phosphatidylinositol 3-kinase. Mutation of IP3R serine 2681 in the Akt substrate motif to alanine (S2681A) or glutamate (S2681E) prevented IP3R phosphorylation in COS cells transfected with constitutively active Akt kinase. Analysis of the Ca2+ flux properties of these IP3R mutants expressed in COS cell microsomes or in DT40 triple knock-out (TKO) cells did not reveal any modification of channel function. However, staurosporine-induced caspase-3 activation in DT40 TKO cells stably expressing the S2681A mutant was markedly enhanced when compared with wild-type or S2681E IP3Rs. We conclude that IP3 receptors are in vivo substrates for Akt kinase and that phosphorylation of the IP3R may provide one mechanism to restrain the apoptotic effects of calcium.     

The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor

A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP(3)R) isoform (type-I long, amino acids1314-1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693-1733 deleted) were expressed as glutathione S-transferase fusion proteins. These domains encompass a putative calmodulin (CaM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CaM in a Ca(2+)-dependent manner as measured by CaM-Sepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CaM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. Full-length cDNAs encoding type-I long, type-I short, and type-III IP(3)R isoforms were expressed in COS cells, and the Ca(2+) sensitivity of [(3)H]IP(3) binding to permeabilized cells was measured. The type-I long isoform was more sensitive to Ca(2+) inhibition (IC(50) = 0.55 microM) than the type-I short (IC(50) = 5.7 microM) or the type-III isoform (IC(50) = 3 microM). In agreement with studies on the fusion proteins, the full-length type-I short bound more CaM-Sepharose, and this binding was inhibited to a greater extent by protein kinase A phosphorylation than the type-I long IP(3)R. Although type-III IP(3)Rs did not bind directly to CaM-Sepharose, hetero-oligomers of type-I/III IP(3)Rs retained the ability to interact with CaM. We conclude that the deletion of the SII splice site in the type-I IP(3)R results in the differential regulation of the alternatively spliced isoforms by Ca(2+), CaM, and protein kinase A.     

Direct association of ligand-binding and pore domains in homo- and heterotetrameric inositol 1,4,5-trisphosphate receptors

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of intracellular Ca(2+) channels that exist as homo- or heterotetramers. In order to determine whether the N-terminal ligand-binding domain is in close physical proximity to the C-terminal pore domain, we prepared microsomal membranes from COS-7 cells expressing recombinant type I and type III IP(3)R isoforms. Trypsin digestion followed by cross-linking and co-immunoprecipitation of peptide fragments suggested an inter-subunit N- and C-terminal interaction in both homo- and heterotetramers. This observation was further supported by the ability of in vitro translated C-terminal peptides to interact specifically with an N-terminal fusion protein. Using a (45)Ca(2+) flux assay, we provide functional evidence that the ligand-binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP(3)Rs and propose that the gating mechanism of IP(3)R Ca(2+) channels involves the association of the N-terminus of one subunit with the C-terminus of an adjacent subunit in both homo- and heterotetrameric complexes.     

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.     

Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP(3)R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP(3)R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP(3)R blockers, reduced Ca(2+) wave activation and global intracellular Ca(2+) ([Ca(2+)](i)) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP(3)R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP(3)R1 being the most abundant isoform at 82% of total IP(3)R message. IP(3)R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca(2+) wave frequency and global [Ca(2+)](i) but abolished UTP-induced Ca(2+) wave activation and reduced the UTP-induced global [Ca(2+)](i) elevation by approximately 61%. Antibodies targeting IP(3)R1 and IP(3)R1 knockdown reduced UTP-induced nonselective cation current (I(cat)) activation. IP(3)R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca(2+) by approximately 45%. These data indicate that IP(3)R1 is the predominant IP(3)R isoform expressed in rat cerebral artery smooth muscle cells. IP(3)R1 stimulation contributes to UTP-induced I(cat) activation, Ca(2+) wave generation, global [Ca(2+)](i) elevation, and vasoconstriction. In addition, IP(3)R1 activation constricts cerebral arteries in the absence of SR Ca(2+) release by stimulating plasma membrane I(cat).     

The type III inositol 1,4,5-trisphosphate receptor is associated with aggressiveness of colorectal carcinoma

The inositol 1,4,5-trisphosphate receptor (InsP3R) mediates Ca(2+) signaling in epithelia and regulates cellular functions such as secretion, apoptosis and cell proliferation. Loss of one or more InsP3R isoform has been implicated in disease processes such as cholestasis. Here we examined whether gain of expression of InsP3R isoforms also may be associated with development of disease. Expression of all three InsP3R isoforms was evaluated in tissue from colorectal carcinomas surgically resected from 116 patients. Type I and II InsP3Rs were seen in both normal colorectal mucosa and colorectal cancer, while type III InsP3R was observed only in colorectal cancer. Type III InsP3R expression in the advancing margins of tumors correlated with depth of invasion, lymph node metastasis, liver metastasis, and TNM stage. Heavier expression of type III InsP3R also was associated with decreased 5-year survival. shRNA knockdown of type III InsP3R in CACO-2 colon cancer cells enhanced apoptosis, while over-expression of the receptor decreased apoptosis. Thus, type III InsP3R becomes expressed in colon cancer, and its expression level is directly related to aggressiveness of the tumor, which may reflect inhibition of apoptosis by the receptor. These findings suggest a previously unrecognized role for Ca(2+) signaling via this InsP3R isoform in colon cancer.     

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP(3)R), each of which has a distinct effect on Ca(2+) signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca(2+)-mediated events. To investigate this question, we examined the effects of each InsP(3)R isoform on transmission of Ca(2+) signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP(3)R isoforms. ATP-induced cytosolic Ca(2+) signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca(2+) signals in mitochondria, which were inhibited more effectively by silencing the type III InsP(3)R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP(3)R. These findings provide evidence that the type III isoform of the InsP(3)R plays a special role in induction of apoptosis by preferentially transmitting Ca(2+) signals into mitochondria.     

Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel playing a major role in Ca2+ signaling. Three isoforms of IP3R have been identified and most cell types express different proportions of each isoform. The DT40 B lymphocyte cell line lacking all three IP3R isoforms (DT40IP3R-KO cells) represents an excellent model to re-express any recombinant IP3R and analyze its specific properties. In the study presented here, we confirmed that DT40IP3R-KO cells do not express any IP3-sensitive Ca2+ release channel. However, with an immunoblot approach and a [3H]IP3 binding approach we demonstrated the presence of a C-terminally truncated form of IP3R type III in the cytosolic fraction of DT40IP3R-KO cells. We further showed that this truncated IP3R retained the ability to couple to the Ca2+ entry channel TRPC6. Therefore, a word of caution is offered about the interpretation of results obtained in using DT40IP3R-KO cells to study the cellular mechanisms of Ca2+ entry.     

The type III inositol 1,4,5-trisphosphate receptor is associated with aggressiveness of colorectal carcinoma

The inositol 1,4,5-trisphosphate receptor (InsP3R) mediates Ca²⁺ signaling in epithelia and regulates cellular functions such as secretion, apoptosis and cell proliferation. Loss of one or more InsP3R isoform has been implicated in disease processes such as cholestasis. Here we examined whether gain of expression of InsP3R isoforms also may be associated with development of disease. Expression of all three InsP3R isoforms was evaluated in tissue from colorectal carcinomas surgically resected from 116 patients. Type I and II InsP3Rs were seen in both normal colorectal mucosa and colorectal cancer, while type III InsP3R was observed only in colorectal cancer. Type III InsP3R expression in the advancing margins of tumors correlated with depth of invasion, lymph node metastasis, liver metastasis, and TNM stage. Heavier expression of type III InsP3R also was associated with decreased 5-year survival. shRNA knockdown of type III InsP3R in CACO-2 colon cancer cells enhanced apoptosis, while over-expression of the receptor decreased apoptosis. Thus, type III InsP3R becomes expressed in colon cancer, and its expression level is directly related to aggressiveness of the tumor, which may reflect inhibition of apoptosis by the receptor. These findings suggest a previously unrecognized role for Ca²⁺ signaling via this InsP3R isoform in colon cancer.     

Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.     

Role of IP₃ receptor in development

IP? receptor is a Ca(2+) release channel localized on the endoplasmic reticulum. IP(3) receptor is composed of three isoforms, which are expressed in various cells and tissues, and play variety of roles throughout development. I here describe the role of IP? receptor from oogenesis, meiotic maturation and fertilization. I also describe the Ca(2+) signaling at meiosis and mitosis, and especially the role in early embryogenesis to determine dorso-ventral axis formation. Loss of function mutation of type 1 IP? receptor in mouse, both by gene targeting and spontaneous mutations shows severe ataxia and other phenotypes. Interestingly, double knockouts of type 1 and type 2 exhibit cardiogenesis arrest and that of type 2 and type 3 results in exocrine secretion deficit. IP?R of Drosophila or Caenorhabditis elegans is single gene and mutation results severe phenotype of behavior. All the data described here show that IP?Rs are essential for life and abnormality of IP(3)Rs results in severe abnormality in its structure and function of organism.     

Effects of chromogranin expression on inositol 1,4,5-trisphosphate-induced intracellular Ca2+ mobilization

We show here that expression of chromogranins in non-neuroendocrine NIH3T3 cells significantly increased the amount of IP(3)-mediated intracellular Ca(2+) mobilization in these cells, whereas suppression of them in neuroendocrine PC12 cells decreased the amount of mobilized Ca(2+). We have therefore investigated the relationship between the IP(3)-induced intracellular Ca(2+) mobilization and secretory granules. The level of IP(3)-mediated Ca(2+) release in CGA-expressing NIH3T3 cells was 40% higher than in the control cells, while that of CGB-expressing cells was 134% higher, reflecting the number of secretory granules formed. Suppression of CGA and CGB expression in PC12 cells resulted in 41 and 78% reductions in the number of secretory granules, respectively, while the extents of IP(3)-induced Ca(2+) release in these cells were reduced 40 and 69%, respectively. The newly formed secretory granules of NIH3T3 cells contained all three isoforms of the IP(3)Rs. Comparison of the concentrations of the IP(3)R isoforms expressed in the ER and nucleus of chromogranin-expressing and nonexpressing NIH3T3 cells did not show significant differences, indicating that chromogranin expression did not affect the expression of endogenous IP(3)Rs. Nonetheless, the IP(3)R concentrations in secretory granules of chromogranin-expressing NIH3T3 cells were 3.5-4.7-fold higher than those of the ER, similar to the levels found in secretory granules of neuroendocrine chromaffin cells, thus suggesting that the IP(3)Rs targeted to the newly formed secretory granules are newly induced by chromogranins without affecting the expression of intrinsic IP(3)Rs. These results strongly suggest that the extent of IP(3)-induced intracellular Ca(2+) mobilization in secretory cells is closely related to the number of secretory granules.     

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.     

Presence of the inositol 1,4,5-triphosphate receptor isoforms in the nucleoplasm

Although the inositol 1,4,5-triphosphate (IP(3))-induced nuclear Ca(2+) release has been shown to play key roles in nuclear functions, the presence of IP(3) receptor (IP(3)R)/Ca(2+) channels in the nucleoplasm has not been found. Recently, the IP(3)R/Ca(2+) channels were reported to exist in the nucleoplasmic reticulum structure, an extension of the nuclear envelope. Here we investigated the potential existence of the IP(3)Rs in the nucleoplasm and found the presence of all three IP(3)R isoforms in neuroendocrine and non-neuroendocrine cells. The IP(3)Rs were widely scattered in the nucleoplasm, localizing in both the heterochromatin and euchromatin regions.     

The spatial distribution of inositol 1,4,5-trisphosphate receptor isoforms shapes Ca2+ waves

Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.     

Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.     

Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.     

Protein 4.1N does not interact with the inositol 1,4,5-trisphosphate receptor in an epithelial cell line

Cytosolic Ca2+ regulates a variety of cell functions, and the spatial patterns of Ca2+ signals are responsible in part for the versatility of this second messenger. The subcellular distribution of the inositol 1,4,5-trisphosphate receptor (IP3R) is thought to regulate Ca2+-signaling patterns but little is known about how the distribution of the IP3R itself is regulated. Here we examined the relationship between the IP3R and the cytoskeletal linker protein 4.1N in the polarized WIF-B cell line because protein 4.1N regulates targeting of the type I IP3R in neurons, but WIF-B cells do not express this cytoskeletal protein. WIF-B cells expressed all three isoforms of the IP3R, and each isoform was distributed throughout the cell. These cells did not express the ryanodine receptor. Photorelease of microinjected, caged IP3 induced a rapid rise in cytosolic Ca2+, but the increase began uniformly throughout the cell rather than at a specific initiation site. Expression of protein 4.1N was not associated with redistribution of the IP3R or changes in Ca2+-signaling patterns. These findings are consistent with the hypothesis that the subcellular distribution of IP3R isoforms regulates the formation of Ca2+ waves, and the finding that interactions between protein 4.1N and the IP3R vary among cell types may provide an additional, tissue-specific mechanism to shape the pattern of Ca2+ waves.