Interactions of Inositol 1,4,5-Trisphosphate (IP3) Receptors with Synthetic Poly(ethylene glycol)-linked Dimers of IP3 Suggest Close Spacing of the IP3-binding Sites

Abstract

The distances between the inositol 1,4,5-trisphosphate (IP 3 )-binding sites of tetrameric IP 3 receptors were probed using dimers of IP 3 linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated 45 Ca 2؉ release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP 3 receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3-4-fold more potent than IP 3 . In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP 3 . The affinity of monomeric IP 3 with only the PEG attached was 2-fold weaker than IP 3 , confirming that the increased affinity of the dimers requires the presence of both IP 3 motifs. The increased affinity of the long dimer probably results from the linked IP 3 molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP 3 to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP 3 to purified receptors. IP 3 and the IP 3 dimers, irrespective of their length, bound with similar affinity to a monomeric IP 3 -binding domain of the type 1 IP 3 receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP 3 -binding sites of an IP 3 receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 ؋ 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP 3 -binding sites of the IP 3 receptor.Inositol 1,4,5-trisphosphate (IP 3 ) 1 receptors are intracellular Ca 2ϩ channels that are expressed in many cells and that mediate the release of Ca 2ϩ from intracellular stores evoked by receptors that stimulate IP 3 formation. The three subtypes of mammalian IP 3 receptor are closely related to each other and to the receptors expressed in birds, Xenopus, crayfish, Drosophila and Caenorhabditis elegans (2). For each of these receptors, the functional IP 3 -gated Ca 2ϩ channel is thought to be a tetramer, which may either be homomeric or, in those species that express more than one receptor subtype, heteromeric (3). Although the different subtypes of mammalian IP 3 receptor differ in their distribution (2), differ modestly in their affinity for IP 3 and their ability to recognize different inositol phosphates (4), and may be differentially modulated (5), the physiological significance of this diversity is unclear. More striking than the differences between IP 3 receptor subtypes are the similarities: the primary sequences of the subunits are closely related, each assembles to form a tetrameric IP 3 -gated Ca 2ϩ channel, they recognize similar ligands with broadly similar affinities, and most are biphasically regulated by cytosolic Ca 2...

Structure of the Type 1 Inositol 1,4,5-Trisphosphate Receptor Revealed by Electron Cryomicroscopy

“…However, our localization is different from that of another electron microscopy study, which suggested the InsP 3 -binding domain is close to the 4-fold axis (8). Their conclusion was inferred from an earlier ligand binding study using dimers of InsP 3 linked by polyethylene glycol molecules of varying lengths (10 -80 Å) (28) that suggested that the InsP 3 -binding sites are separated by no more than 20 Å. This estimate is inconsistent with our map where the InsP 3 -binding sites assigned to the B domains are spaced at least 100 Å apart.…”

“…This estimate is inconsistent with our map where the InsP 3 -binding sites assigned to the B domains are spaced at least 100 Å apart. A possible explanation for this discrepancy is that the radioligand binding studies were performed at pH 8.3 (28), which is optimal for binding of InsP 3 , while our study and the heparin-binding site mapping study (26) used a pH of 7.4. Conformational transitions may take place in the channel upon changing the pH as suggested previously (27).…”

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

“…Our 3D structure (determined in the absence of Ca 2ϩ ), which measures about 18 ϫ 18 ϫ 18 nm and is approximately square in both top and side projections, corresponds more closely with the square structure observed by Hamada et al (31). However, the peripheral location of the IP 3 -binding domain suggested by heparin-gold labeling (31) is not consistent with recent studies (32) suggesting that the IP 3 -binding sites within a tetrameric receptor are no more than 2 nm apart. It is possible that the large size of the heparin, gold, and linker may have exaggerated the separation of the IP 3 -binding sites in the labeling study (31).…”

“…Results from both limited proteolysis and measurements of the location and properties of the IP 3 -binding sites can be used as guides to assign these domains. The tetrameric IP 3 R has a single IP 3 -binding site on each subunit, located between residues 226 and 576 (9), and recent studies with dimeric molecules of IP 3 suggest that the sites are likely to be separated by no more than Ϸ2 nm (32). On this basis, the petal subdomains in our 3D map are unlikely to contain the IP 3 -binding sites, because equivalent sites within these subdomains are at least 8 nm apart (Fig.…”

Counting Functional Inositol 1,4,5-Trisphosphate Receptors into the Plasma Membrane

“…The His 6 -tagged fusion proteins were expressed in Escherichia coli (19) and cleaved from their His 6 tags using thrombin. Equilibrium-competition binding assays using 3 H-IP 3 (0.7 nM) were performed and analyzed as reported previously (19).IP 3 -evoked Ca 2ϩ Release-The ER of DT40 cells was loaded with a low affinity Ca 2ϩ indicator (Mag fluo-4), and IP 3 -evoked Ca 2ϩ release was measured from the saponin-permeabilized cells using a FlexStation as previously reported (20).Single Channel Recording-Single channel currents were recorded in the whole-cell configuration or from excised patches of nuclear envelope using the patch clamp technique exactly as reported (10). Except where indicated otherwise, bath solution (BS) contained 140 mM KCl, 10 mM Hepes, 500 M BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid), 270 M CaCl 2 (free [Ca 2ϩ ] ϭ 246 nM), pH 7.1, and pipette solution contained: 140 mM KCl, 10 mM Hepes, 100 M BAPTA, 48.7 M CaCl 2 (free [Ca 2ϩ ] ϭ 212 nM), 500 M ATP, pH 7.1, and IP 3 (usually 10 M).…”