Single-channel function of recombinant type 2 inositol 1,4, 5-trisphosphate receptor

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.     

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor

High affinity Ins(1,4,5)P₃-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins91,4,5)P₃ on intracellular Ca²⁺ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized bind Ins(1,4,5)P₃ with an affinity (K_d = 7.26 ± 0.52 nM, Hill coefficient H = 1.05 ± 0.06) similar to that of the sites in native membranes (K_d = 6.02 ± 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P₃ Ins(4,5)P₂, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP₃ receptor will allow its further characterization, purification, and comparison of its properties with those of InsP₃ receptors already purified from cerebellum and smooth muscle.     

Hormonal regulation of inositol 1,4,5-trisphosphate receptor in rat liver

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from an intracellular store. We have investigated the properties of the [32P]IP3 binding sites in rat liver. Two specific [32P]IP3 receptors with KD of 2.3 and 88 nM and respective capacities of 33 fmol/mg protein and 195 fmol/mg protein have been detected in a crude membrane fraction prepared from rat liver homogenate. The pretreatment of the liver with IP3-dependent hormones increased two-fold the capacity of the high affinity site. This effect was partly reversed by dibutyryl cyclic AMP. Permeabilized hepatocytes also displayed two [32P]IP3 binding sites with KD of 1.5 and 84 nM and respective capacities of 8 and 300 fmol/10(6) cells. We have measured the [32P]IP3 binding and the IP3-induced 45Ca2+ release in the same batch of permeabilized hepatocytes. In a low Mg2+ medium, the EC50 for 45Ca2+ release was in close correlation with the KD for the low affinity site. These data suggest that an equilibrium between two states of the IP3 receptor is regulated by hormone action and the low affinity state is responsible for the intracellular Ca2+ release.     

Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2-aminoethoxydiphenylborate

2-Aminoethoxydiphenylborate (2-APB) inhibits the extent of inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release from cerebellar microsomes with a potency that is dependent upon the InsP(3) concentration used. At high InsP(3) concentrations (10 microM), the concentration of 2-APB required to cause half-maximal InsP(3)-induced Ca(2+) release (IC(50)) was greater than 1 mM, while at 0.25 microM InsP(3) this reduced to 220 microM. The fact that the inhibition of the extent of InsP(3)-induced Ca(2+) release (IICR) by 2-APB was not restored to control levels by high concentrations of InsP(3), in addition to the fact 2-APB did not substantially inhibit [3H]InsP(3) binding to its receptor, indicates that the inhibition is not competitive in nature. Since the cooperativity of IICR as a function of InsP(3) was reduced in the presence of 2-APB (Hill coefficient changing from 1.9 in the absence of 2-APB to 1.4 in the presence of 1 mM 2-APB), this suggests that it is acting as an allosteric inhibitor. 2-APB also reduces the rate constants for IICR. In cerebellar microsomes this release process is biphasic in nature, with a fast and slow phase. 2-APB appears particularly to affect the fast-phase component. Although 2-APB does not inhibit the ryanodine receptor, it does inhibit the Ca(2+) ATPase activity as well store-operated Ca(2+) entry channels, which may limit its use as a specific membrane permeant InsP(3) receptor inhibitor.     

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.     

The inositol 1,4,5-trisphosphate receptor (Itpr) gene family in Xenopus: identification of type 2 and type 3 inositol 1,4,5-trisphosphate receptor subtypes

Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the 'Xenopus IP3R' cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.     

4.1N binding regions of inositol 1,4,5-trisphosphate receptor type 1

Zhang et al. and Maximov et al. [S. Zhang, A. Mizutani, C. Hisatsune, T. Higo, H. Bannai, T. Nakayama, M. Hattori, and K. Mikoshiba, Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells, J. Biol. Chem. 278 (2003) 4048-4056; A. Maximov, T. S. Tang, and I. Bezprozvanny, Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons, Mol. Cell. Neurosci. 22 (2003) 271-283.] reported that 4.1N is a binding partner of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1), however the binding site of IP(3)R1 differed: the former determined the C-terminal 14 amino acids of the cytoplasmic tail (CTT14aa) as the binding site, while the latter assigned another segment, cytoplasmic tail middle 1 (CTM1). To solve this discrepancy, we performed immunoprecipitation and found that both the segments had binding activity to 4.1N. Both segments also interfered the 4.1N-regulated IP(3)R1 diffusion in neuronal dendrites. However, IP(3)R1 lacking the CTT14aa (IP(3)R1-DeltaCTT14aa) does not bind to 4.1N [S. Zhang, A. Mizutani, C. Hisatsune, T. Higo, H. Bannai, T. Nakayama, M. Hattori, and K. Mikoshiba, Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells, J. Biol. Chem. 278 (2003) 4048-4056.] and its diffusion constant is larger than that of IP(3)R1 full-length in neuronal dendrites [K. Fukatsu, H. Bannai, S. Zhang, H. Nakamura, T. Inoue, and K. Mikoshiba, Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites, J. Biol. Chem. 279 (2004) 48976-48982.]. We conclude that both the CTT14aa and CTM1 sequences can bind to 4.1N in peptide fragment forms. However, we propose that the responsible binding site for 4.1N binding in full-length tetramer form of IP(3)R1 is CTT14aa.     

Mass spectrometric analysis of type 1 inositol 1,4,5-trisphosphate receptor ubiquitination

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP(3)-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP(3) levels, IP(3) receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP(3) receptor (IP(3)R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates. Our findings are (i) that at least 11 of the 167 lysines in IP(3)R1 can be ubiquitinated and that these are clustered in the regulatory domain and are found in surface regions, (ii) that at least approximately 40% of the IP(3)R1-associated ubiquitin is monoubiquitin, (iii) that both Lys(48) and Lys(63) linkages are abundant in attached ubiquitin chains, and (iv) that Lys(63) linkages accumulate most rapidly. Additionally, we find that not all IP(3)R1 subunits in a tetramer are ubiquitinated and that nontetrameric IP(3)R1 complexes form as degradation proceeds, suggesting that ubiquitinated subunits may be selectively extracted and degraded. Overall, these data show that endogenous IP(3)R1 is tagged with an array of ubiquitin conjugates at multiple sites and that both IP(3)R1 ubiquitination and degradation are highly complex processes.     

Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP(3)R) results in a more pronounced Ca(2+)release in the presence of inositol 1,4,5-trisphosphate (IP(3)). Two recently published studies demonstrated a different ATP sensitivity of IP(3)-induced Ca(2+)release in cell types expressing different IP(3)R isoforms. Cell types expressing mainly IP(3)R3 were less sensitive to ATP than cell types expressing mainly IP(3)R1 (Missiaen L, Parys JB, Sienaert I et al. Functional properties of the type 3 InsP(3)receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem 1998;273: 8983-8986; Miyakawa T, Maeda A, Yamazawa T et al. Encoding of Ca(2+)signals by differential expression of IP(3)receptor subtypes. EMBO J 1999;18: 1303-1308). In order to investigate the difference in ATP sensitivity between IP(3)R isoforms at the molecular level, microsomes of Sf9 insect cells expressing full-size IP(3)R1 or IP(3)R3 were covalently labeled with ATP by using the photoaffinity label 8-azido[alpha-(32)P]ATP. ATP labeling of the IP(3)R was measured after immunoprecipitation of IP(3)Rs with isoform-specific antibodies, SDS-PAGE and Phosphorimaging. Unlabeled ATP inhibited covalent linking of 8-azido[alpha-(32)P]ATP to the recombinant IP(3)R1 and IP(3)R3 with an IC(50)of 1.6 microM and 177 microM, respectively. MgATP was as effective as ATP in displacing 8-azido[alpha-(32)P]ATP from the ATP-binding sites on IP(3)R1 and IP(3)R3, and in stimulating IP(3)-induced Ca(2+)release from permeabilized A7r5 and 16HBE14o- cells. The interaction of ATP with the ATP-binding sites on IP(3)R1 and IP(3)R3 was different from its interaction with the IP(3)-binding domains, since ATP inhibited IP(3)binding to the N-terminal 581 amino acids of IP(3)R1 and IP(3)R3 with an IC(50)of 353 microM and 4.0 mM, respectively. The ATP-binding sites of IP(3)R1 bound much better ATP than ADP, AMP and particularly GTP, while IP(3)R3 displayed a much broader nucleotide specificity. These results therefore provide molecular evidence for a differential regulation of IP(3)R1 and IP(3)R3 by ATP.     

Characterization of inositol 1,3,4-trisphosphate phosphorylation in rat liver

Liver homogenates phosphorylated Ins 1,3,4-P3 to an InsP4 isomer that was distinct from Ins 1,3,4,5-P4. This InsP4 isomer accumulated in vasopressin stimulated hepatocytes prelabeled with myo-[3H]inositol with a time course that lagged behind Ins 1,3,4-P3 formation. The Ins 1,3,4-P3 kinase responsible for its formation was partially purified from rat liver. The enzyme had a Km for Ins 1,3,4-P3 of 0.29 microM, a Km for ATP of 141 microM and was not affected by changes in free Ca2+ in the physiological range. The relationship of this new InsP4 isomer to the inositol phosphate signaling pathway is discussed.     

Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling

Ca(2+) is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca(2+) signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca(2+) when administrated alone, becomes capable of evoking [Ca(2+)](i) increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP(3)R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP(3)R complex functions as a focal point to promote Ca(2+) release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP(3)R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP(3)R1 at the Ca(2+) store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP(3)R complex connects different signaling pathways to define the fidelity and specificity of Ca(2+) signaling.     

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.     

Differential expression of inositol 1,4,5-trisphosphate receptor types 1, 2, and 3 in rat myometrium and endometrium during gestation

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF(4)(-) was enhanced with advancing gestation. (3)[H]IP(3) binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP(3)R as well as their corresponding proteins, IP(3)R-1, IP(3)R-2, and IP(3)R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP(3)Rs increased with advancing gestation, except for IP(3)R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP(3)Rs. The difference noted between the two uterine tissues suggests that IP(3)Rs may have cell-specific functions.     

Structure and expression of the rat inositol 1,4,5-trisphosphate receptor

The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.     

Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytes

Inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1) is already known to be highly expressed in the brain, and is found in many other tissues, including the atrium of the heart. Although the complete primary structure of IP₃R1 in the rat brain has been reported, the complete sequence of an IP₃R1 clone from atrial myocytes has not been reported. We isolated an IP₃R1 complementary DNA (cDNA) clone from isolated adult rat atrial myocytes, and found a new splice variant of IP₃R1 that was different from a previously reported IP₃R1 cDNA clone obtained from a rat brain (NCBI GenBank accession number: NM_001007235). Our clone had 99% similarity with the rat brain IP₃R1 sequence; the exceptions were 39 amino acid deletions at the position of 1693–1731, and the deletion of phenylalanine at position 1372 that lay in the regulatory region. Compared with the rat brain IP₃R1, in our clone proline was replaced with serine at residue 2439, and alanine was substituted for valine at residue 2445. These changes lie adjacent to or within the fifth transmembrane domain (2440–2462). Although such changes in the amino acid sequences were different from the rat brain IP3R1 clone, they were conserved in human or mouse IP3R1. We produced a plasmid construct expressing the atrial IP3R1 together with green fluorescent protein (GFP), and successfully overexpressed the atrial IP3R1 in the adult atrial cell line HL-1. Further investigation is needed on the physiological significance of the new splice variant in atrial cell function.     

Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation

Egg activation and further embryo development require a sperm-induced intracellular Ca²⁺ signal at the time of fertilization. Prior to fertilization, the egg's Ca²⁺ machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca²⁺ releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1), which is responsible for most of this Ca²⁺ release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP₃R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP₃R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T²⁶⁵⁶ as a major Plk1 site on IP₃R1. We therefore propose that the initial increase in IP₃R1 sensitivity during oocyte maturation is underpinned by IP₃R1 phosphorylation at an MPM-2 epitope(s).     

Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.     

Structure of the type 1 inositol 1,4,5-trisphosphate receptor revealed by electron cryomicroscopy

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.