[Abstract unavailable

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated Ca(2+) channel that is located on intracellular Ca(2+) stores and modulates Ca(2+) signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP(3)R type 1 (IP(3)R1) to identify IP(3)R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP(3)R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP(3)R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP(3)R1. Biochemical analysis also showed that CARP is co-precipitated with IP(3)R1. Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP(3)R1, and that the CARP-binding site is located within the modulatory domain of IP(3)R1 amino acids 1387-1647. CARP inhibits IP(3) binding to IP(3)R1 by reducing the affinity of the receptor for IP(3). As reported previously, sensitivity to IP(3) for IP(3)-induced Ca(2+) release in Purkinje cells is low compared with that in other tissues. This could be due to co-expression of CARP with IP(3)R in Purkinje cells and its inhibitory effects on IP(3) binding.

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress in vivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.

, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "headto-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP 3 R1 with Ca 2؉ also contains the four bridges connecting from the IP 3 -binding domain toward the channel domain. These data suggest that the Ca 2؉ -specific conformational change structurally regulates the IP 3 -triggered channel opening within IP 3 R1.Inositol 1,4,5-trisphosphate receptor (IP 3 R) 1 acts as a highly controlled decoder transducing extracellular stimuli into intracellular calcium (Ca 2ϩ ) signals, a process that plays an integral role in vital morphogenesis and physiological plasticity (1). IP 3 Rs are structurally divided into three regions: 1) a large cytoplasmic region with an IP 3 binding pocket close to the N terminus; 2) a transmembrane region near the C terminus; and 3) a short C-terminal tail (2). The most characterized IP 3 R, type 1 IP 3 R (IP 3 R1), a predominant type in the cerebellar endoplasmic reticulum (ER) and spine apparatus, is a 2749-amino acid polypeptide (2) containing the IP 3 binding core (residues 226 -578) (3, 4), a transmembrane region composed of putative six-spanning domains (residues 2276 -2589) (5, 6). The transmembranous segments are essential for the tetramerization (7, 8) to form a channel domain as in the case of the voltage-, Ca 2ϩ -, and cyclic nucleotide-gated ion channel superfamily (9, 10).The IP 3 never fully opened the channel of IP 3 R without Ca 2ϩ . Previous functional analysis indicates that the low Ca 2ϩ level acts as an essential co-agonist for IP 3 -gated Ca 2ϩ release (11)(12)(13)(14). The requirement of the dual ligands is considered to be an important basis of the signaling cross-talk involved in the cell function (15). Thus, it would be useful to understand how the binding of co-agonists structurally opens the channel of IP 3 R. Recently, the x-ray structure of the crystallized IP 3 binding core has revealed the spatial relationship among a specific IP 3 -binding site and two plausible Ca 2ϩ -binding sites (4). The local allosteric coupling of IP 3 -and Ca 2ϩ -binding sites in this domain is thought to be one of the possible elements for the requirement of dual agonists to open the channel. However, additional six Ca 2ϩ -binding sites have been detected by extensive screening of expressed IP 3 R1-fragments outside the IP 3 binding core (16,17) and, based on the primary sequence, a critical residue for the Ca 2ϩ -activated channel opening was located far from the IP 3 binding core (18,19). Furthermore, allosteric sites for regulations were widely distributed outside the IP 3 binding core (20). Thus, it is quite important to visualize the overall behavior of tetrameric IP 3 R to elucidate the mechanism underlying channel gating by IP 3 and Ca 2ϩ . Knowledge of the spatial interrelations and rearrangements of the...

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Inositol 1,4,5-trisphosphate receptor (IP 3 R) is a highly controlled calcium (Ca 2؉ ) channel gated by inositol 1,4,5-trisphosphate (IP 3 ). Multiple regulators modulate IP 3 -triggered pore opening by binding to discrete allosteric sites within IP 3 R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca 2؉ , the most pivotal regulator, induced marked structural changes in the tetrameric IP 3 R purified from mouse cerebella. Electron microscopy of the IP 3 R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca 2؉ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP 3 -binding domains, assigned by binding to heparin-gold. Ca 2؉ -dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca 2؉ appeared to regulate IP 3 gating activity through the rearrangement of functional domains.Inositol 1,4,5-trisphosphate receptor (IP 3 R) 1 is a tetrameric ion channel that release Ca 2ϩ from intracellular stores in response to the binding of 1,4,5-trisphosphate (IP 3 ), a second messenger generated by various extracellular stimuli, neurotransmitters, neuromodulators, hormones, and lights (1, 2). The IP 3 R is widely distributed in living systems and plays pivotal roles in fundamental processes including fertilization, cellular proliferation and differentiation, cellular signaling, and vesicle secretion (2). Molecular cloning studies have revealed that there are three isoforms of IP 3 R and that alternative splicing results in several variants of the IP 3 R (2). These divergent primary structures of the IP 3 R and their differential distributions have been assumed to award the functional diversity of IP 3 R by nature.The most characterized type 1 IP 3 R (IP 3 R1), a predominant type in rodent cerebellar endoplasmic reticulum (ER) and spine apparatus, plays an integral role in Ca 2ϩ signaling (3-5) and neural plasticity (6, 7). The protomer of IP 3 R1, a 2749-amino acid polypeptide (M r 313,000), contains the IP 3 -binding core (residues 226 -578), membrane-spanning domains (residues 2276 -2589), and widespread allosteric sites for intracellular effector molecules (Ca 2ϩ , calmodulin, and ATP) and for phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, Ca 2ϩ / calmodulin-dependent protein kinase II, and tyrosine kinase) (2). These cumulative allosteric regulations imply a structural paradigm for global conformational changes within the higher ordered structure of IP 3 R1.Because Ca 2ϩ rigorously determines the channel activity of IP 3 R and Ca 2ϩ -dependent behavior of IP 3 R is considered to be crucial for spatiotemporal organizations of Ca 2ϩ signaling (1, 4), the most important regulator for IP 3 R is Ca 2ϩ per se. Previous functional analysis indicates that a low Ca 2ϩ level acts a...

The inositol 1,4,5-trisphosphate (IP) receptor (IPR) is an IP-gated ion channel that releases calcium ions (Ca) from the endoplasmic reticulum. The IP-binding sites in the large cytosolic domain are distant from the Ca conducting pore, and the allosteric mechanism of how IP opens the Ca channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IPR in the absence and presence of IP Analyses of two distinct space group crystals uncovered an IP-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IPR channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP binding to the Ca channel.

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