Modulation of mammalian inositol 1,4,5-trisphosphate receptor isoforms by calcium: a role of calcium sensor region

In the accompanying article, we compared main functional properties of the three mammalian inositol 1,4,5-trisphosphate receptors (InsP3R) isoforms. In this article we focused on modulation of mammalian InsP3R isoforms by cytosolic Ca2+. We found that: 1), when recorded in the presence of 2 microM InsP3 and 0.5 mM ATP all three mammalian InsP3R isoforms display bell-shaped Ca2+ dependence in physiological range of Ca2+ concentrations (pCa 8-5); 2), in the same experimental conditions InsP3R3 is most sensitive to modulation by Ca2+ (peak at 107 nM Ca2+), followed by InsP3R2 (peak at 154 nM Ca2+), and then by InsP3R1 (peak at 257 nM Ca2+); 3), increase in ATP concentration to 5 mM had no significant effect of Ca2+ dependence of InsP3R1 and InsP3R2; 4), increase in ATP concentration to 5 mM converted Ca2+ dependence of InsP3R3 from "narrow" shape to "square" shape; 5), ATP-induced change in the shape of InsP3R3 Ca2+ dependence was mainly due to an >200-fold reduction in the apparent affinity of the Ca2+-inhibitory site; 6), the apparent Ca2+ affinity of the Ca2+ sensor region (Cas) determined in biochemical experiments is equal to 0.23 microM Ca2+ for RT1-Cas, 0.16 microM Ca2+ for RT2-Cas, and 0.10 microM Ca2+ for RT3-Cas; and 7), Ca2+ sensitivity of InsP3R1 and InsP3R3 isoforms recorded in the presence of 2 microM InsP3 and 0.5 mM ATP or 2 microM InsP3 and 5 mM ATP can be exchanged by swapping their Cas regions. Obtained results provide novel information about functional properties of mammalian InsP3R isoforms and support the importance of the Ca2+ sensor region (Cas) in determining the sensitivity of InsP3R isoforms to modulation by Ca2+.     

Distribution profile of inositol 1,4,5-trisphosphate receptor isoforms in adrenal chromaffin cells

Given the importance of inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels in the control of intracellular Ca(2+) concentrations, we determined the relative concentrations of the IP(3)R isoforms in subcellular organelles, based on serially sectioned electron micrographs. The endoplasmic reticulum (ER) was estimated to contain 15-20% of each of the three IP(3)R isoforms while secretory granules contained 58-69%. The nucleus contained approximately 15% each of IP(3)R-1 and -2, but 25% of IP(3)R-3, whereas the plasma membrane contained approximately 1% or less of each. These suggested that secretory granules, the nucleus and ER are at the center of IP(3)-dependent intracellular Ca(2+) control mechanisms in chromaffin cells.     

Molecular characterization of the inositol 1,4,5-trisphosphate receptor pore-forming segment

Specific residues in the putative pore helix, selectivity filter, and S6 transmembrane helix of the inositol 1,4,5-trisphosphate receptor were mutated in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel (C2533A, G2541A, G2545A, G2546A, and G2547A). Of the remaining three mutants, C2527A and R2543A were partially active and G2549A behaved like wild type receptor. Mutation of a putative glycine hinge residue in the S6 helix (G2586A) or a putative gating residue at the cytosolic end of S6 helix (F2592A) had minimal effects on function, although channel function was inactivated by G2586P and F2592D mutations. The mutagenesis data are interpreted in the context of a structural homology model of the inositol 1,4,5-trisphosphate receptor.     

Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants

The inositol (1,4,5)-trisphosphate receptor (InsP(3)R) is an intracellular calcium (Ca(2+)) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP(3)R gene (itpr) encodes a protein (DmInsP(3)R) that is approximately 60% conserved with mammalian InsP(3)Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP(3)Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP(3)R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP(3)R-the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP(3)R (conductance, gating, and sensitivity to InsP(3) and Ca(2+)) are remarkably conserved with the mammalian InsP(3)R1; 2), single-channel conductance of the adult head DmInsP(3)R isoform is 89 pS and the embryonic DmInsP(3)R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP(3)Rs to activation by InsP(3), but not their InsP(3)-binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca(2+); and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP(3)-induced Ca(2+) fluxes in microsomes isolated from wild-type and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP(3)R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP(3)R function.     

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.     

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.     

Functional coupling of chromogranin with the inositol 1,4,5-trisphosphate receptor shapes calcium signaling

Chromogranins A and B are high capacity, low affinity calcium (Ca(2+)) storage proteins that bind to the inositol 1,4,5-trisphosphate-gated receptor (InsP(3) R). Although most commonly associated with secretory granules of neuroendocrine cells, chromogranins have also been found in the lumen of the endoplasmic reticulum (ER) of many cell types. To investigate the functional consequences of the interaction between the InsP(3) R and the chromogranins, we disrupted the interaction between the two proteins by adding a chromogranin fragment, which competed with chromogranin for its binding site on the InsP(3)R. Responses were monitored at the single channel level and in intact cells. When using InsP(3) R type I incorporated into planar lipid bilayers and activated by cytoplasmic InsP(3) and luminal chromogranin, the addition of the fragment reversed the enhancing effect of chromogranin. Moreover, the expression of the fragment in the ER of neuronally differentiated PC12 cells attenuated agonist-induced intracellular Ca(2+) signaling. These results show that the InsP(3)R/chromogranin interaction amplifies Ca(2+) release from the ER and that chromogranin is an essential component of this intracellular channel complex.     

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, 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 "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.     

The inositol 1,4,5-trisphosphate (InsP3) receptor

Conclusion In this review, we have described the functional properties and regulation of the InsP₃R. Not all aspects of InsP₃R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP₃R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP₃R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP₃R, the structural determinants responsible for regulation of InsP₃R by Ca and ATP, the functional effects of InsP₃R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.We thank S. Bezprozvannaya for expert technical assistance. This work was supported by National Institutes of Health grants HL 33026 and GM 39029, and a Grant-in-Aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.     

Modelling the effect of specific inositol 1,4,5-trisphosphate receptor isoforms on cellular Ca2+ signals

BACKGROUND INFORMATION: Oscillations of cytosolic Ca2+ are well-known to rely on the regulatory properties of the InsP3R (inositol 1,4,5-trisphosphate receptor). Three isoforms of this channel have been identified. They differ in their regulatory properties by Ca2+ and InsP3. Experiments in different cell types clearly indicate that the relative amounts of each isoform affect the time course of Ca2+ changes after agonist stimulation. In the present study, we investigate whether different steady-state curves for the open probability of the InsP3Rs as a function of Ca2+ imply different dynamical behaviours when these receptors are present in a cellular environment. We therefore describe by a specific phenomenological model the three main types of curves that have been reported: (i) the classical bell-shaped curve, (ii) the bell-shaped curve that is shifted towards higher Ca2+ concentrations when InsP3 is increased, and (iii) a monotonous increasing function of cytosolic Ca2+. RESULTS: We show that, although these types of curves can be ascribed to slight differences in the channel regulation by Ca2+ and InsP3, they can indicate important variations as to the receptor role in cellular Ca2+ control. Thus the receptor associated with the classical bell-shaped curve appears to be the most robust Ca2+ oscillator. If the steady-state curve is supposed to be a monotonous increasing function of cytosolic Ca2+, the modelled receptor cannot sustain Ca2+ oscillations in the absence of Ca2+ exchanges with the extracellular medium. When the bell-shaped curve is shifted towards higher Ca2+ concentrations with increasing InsP3 levels, the model predicts that the receptor is less robust to changes in density; this receptor, however, provides a finer control of the steady-state level of Ca2+ when varying the InsP3 concentration. CONCLUSIONS: Our model allows us to propose an explanation for the experimental observations about the effect of selectively expressing or down-regulating InsP3R isoforms, as well as to make theoretical predictions.     

The inositol 1,4,5-trisphosphate receptors

The inositol (1,4,5)-trisphosphate receptors (InsP3R) are the intracellular calcium (Ca2+) release channels that play a key role in Ca2+ signaling in cells. Three InsP3R isoforms-InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals. A single InsP3R isoform is expressed in Drosophila melanogaster (DmInsP3R) and Caenorhabditis elegans (CeInsP3R). The progress made during last decade towards understanding the function and the properties of the InsP3R is briefly reviewed in this chapter. The main emphasis is on studies that revealed structural determinants responsible for the ligand recognition by the InsP3R, ion permeability of the InsP3R, modulation of the InsP3R by cytosolic Ca2+, ATP and PKA phosphorylation and on the recently identified InsP3R-binding partners. The main focus is on the InsP3R1, but the recent information about properties of other InsP3R isoforms is also discussed.     

Studying isoform-specific inositol 1,4,5-trisphosphate receptor function and regulation

Inositol 1,4,5-trisphosphate receptors (InsP3R) are a family of ubiquitously expressed intracellular Ca2+ channels. Isoform-specific properties of the three family members may play a prominent role in defining the rich diversity of the spatial and temporal characteristics of intracellular Ca2+ signals. Studying the properties of the particular family members is complicated because individual receptor isoforms are typically never expressed in isolation. In this article, we discuss strategies for studying Ca2+ release through individual InsP3R family members with particular reference to methods applicable following expression of recombinant InsP3R and mutant constructs in the DT40-3KO cell line, an unambiguously null InsP3R expression system.     

Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum.

The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsP3 receptor protein is composed of four noncovalently bound identical subunits each with a Mr of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2(+)-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [alpha-32P]8-azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the Bmax and Kd values for ATP binding of 2.3 pmol/micrograms and 17 microM, respectively. Reconstitution of the purified InsP3 receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 mM Ca2+) and sodium conductance (21 pS in 100-500 mM asymmetric Na+ solutions) with permeability ratios of PCa/PTris = 6.3 and PNa/PCl = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.     

Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster.

We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP3R) of Drosophila melanogaster. The predicted Drosophila InsP3R (2,833 amino acids) has extensive sequence similarity to the mouse InsP3R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP3-binding activity. The Drosophila InsP3R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP3R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP3R in visual and olfactory transduction. In addition, the InsP3R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [3H]InsP3 binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP3/Ca2+ signaling system in Drosophila.     

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).     

Immunohistochemical localization of type 2 inositol 1,4,5-trisphosphate receptor to the nucleus of different mammalian cells

The inositol 1,4,5-trisphosphate receptor (InsP3R) is a ligand-gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti-InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS-7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co-localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity-binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed.     

Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

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.     

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.     

Carboxyl-terminal sequences critical for inositol 1,4,5-trisphosphate receptor subunit assembly

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of conserved subunits that each contains six transmembrane regions (TMRs) localized near the carboxyl terminus. Receptor subunit assembly into a tetramer appears to be a multideterminant process involving an additive contribution of membrane spanning helices and the short cytosolic carboxyl terminus (residues 2590-2749). Previous studies have shown that of the six membrane-spanning regions in each subunit, the 5th and 6th transmembrane regions, and the carboxyl terminus are strong determinants for assembly. The fifth and sixth TMRs contain numerous beta-branched amino acids that may participate in coiled/coil formation via putative leucine zipper motifs. InsP(3)R truncation mutants were expressed in COS-1 cells and analyzed by sucrose density gradient sedimentation and gel filtration for their ability to assemble. Chemical cross-linking with the homobifunctional reagents sDST or DMS of mammalian and bacterially expressed carboxyl-terminal containing receptor fragments reveals that sequences within the carboxyl terminus confer the formation of subunit dimers. A series of InsP(3) receptor carboxyl-terminal fragments and glutathione S-transferase (GST)/InsP(3)R chimeras were expressed in Escherichia coli and used in an in vitro assay to elucidate the minimal sequence responsible for association of the carboxyl termini into dimers. The results presented here indicate that this minimal sequence is approximately 30 residues in length and is localized between residues 2629 and 2654. These residues are highly conserved between the three InsP(3)R isoforms ( approximately 80% identity) as well as the ryanodine receptor ( approximately 40% identity) and suggest that a conserved assembly motif may exist between the two intracellular receptor families. We propose that assembly of the InsP(3) receptor to a tetramer involves intersubunit interactions mediated through both the membrane-spanning regions and residues 2629-2654 of the carboxyl terminus possibly through the formation of a dimer of dimers.