Carboxyl-terminal Sequences Critical for Inositol 1,4,5-Trisphosphate Receptor Subunit Assembly

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The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP 3 R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP 3 -and heparin-sensitive channels with a unitary conductance similar to native InsP 3 Rs. 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 InsP 3 -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 InsP 3 R1 tetramer.The inositol 1,4,5-trisphosphate receptor (InsP 3 R) 1 is a ligand-gated ion channel that modulates Ca 2ϩ release from intracellular stores. The InsP 3 R is widely distributed in metazoans and plays a key role in diverse physiological functions. In mammals, three different InsP 3 R isoforms are expressed; each is encoded by a distinct gene. The three isoforms are homologous and share ϳ70% amino acid identity. Functional channels are composed of four subunits that tetramerize via determinants localized within the transmembrane regions and the cytosolic C terminus (1-3). Individual cell types often express more than one isoform, which may be present as homo-or heterotetrameric populations. The most characterized type 1 InsP 3 R (InsP 3 R1) forms largely homotetramers in cerebellum with M r of 313,000 (2749 residues) for the monomer. Based on biochemical, molecular biological, and electrophysiological studies, each monomer subunit of the InsP 3 R1 is organized into three principal functional regions: an N-terminal InsP 3 -binding region (residues 226 -578), a central (ϳ1,700-residue) modulatory/coupling region, and a channel-forming region near the C terminus. The channel region contains six transmembrane segments interspersed within residues 2276 -2590 that are critical for membrane targeting, oligomerization, and formation of the ion permeation pathway (1-4). Thus, both ends, the large N-terminal region (residues 1-2275) and the C-terminal region (residues 2590 -2749), are exposed to the cytoplasm.Electron micrographs of the negatively stained detergentsolubilized InsP 3 R1 and freeze-fracture deep etching electron microscopy of endoplasmic reticulum have been used to characterize the morphology of the InsP 3 R1 channel (5, 6). But the three-dimensional shape and dimensions of the channel particles were not determined. Two three-dimensional maps of the InsP 3 R1 were reported recently based on single-particle reconstruction from specimens prepared by ice embedding (7) and negative staining (8). There is a poor agreement between these two maps with respect to the overall appearance and dimensions of the channel structure. In this study, we present a 30-Å three-dimensional s...

Section: Three-dimensional Structure Of Type 1 Insp 3 R 21321supporting

confidence: 87%

“…The three isoforms are homologous and share ϳ70% amino acid identity. Functional channels are composed of four subunits that tetramerize via determinants localized within the transmembrane regions and the cytosolic C terminus (1)(2)(3). Individual cell types often express more than one isoform, which may be present as homo-or heterotetrameric populations.…”

mentioning

confidence: 99%

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

Serysheva

Bare

Ludtke

et al. 2003

Self Cite

“…We examined the selfassociation properties of the wild-type and mutant C-tails in the presence of a noncleavable heterobifunctional cross-linker (SMPH) that cross-links -NH 2 and -SH groups in close proximity. As observed previously, a small fraction of dimers survives the denaturing conditions of SDS-PAGE, and the formation of the dimer bands is enhanced in the presence of the cross-linking agent (39) (Fig. 4D).…”

Section: Gating Residues and Charged Residues In The Distal Portion Osupporting

confidence: 84%

“…A previously documented property of the C-tail is the ability to self-associate to form dimers (39). A coiled-coil domain has also been identified in the C-tail (amino acids 2694 -2721), which forms tetramers in vitro (14).…”

Section: Gating Residues and Charged Residues In The Distal Portion Omentioning

confidence: 99%

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

Bhanumathy

Fonseca

Morris

et al. 2012

Background: Mutagenesis was used to gain insights into the structure/function of IP 3 R channels. Results: Only 5 of 22 residues mutated proved essential for channel function. Conclusion: We propose that Ile-2588 and Ile-2589 are at the channel gate. Arg-2596, His-2630, and His-2635 maintain the structure of the pore and facilitate contacts critical for channel gating. Significance: A revised model of channel gating is proposed.

“…The C terminus of each subunit is postulated to span intracellular membranes six times and forms a single cation-selective pore (13,14). In addition, this region signals retention of the protein to the endoplasmic reticulum (15,16). Although the InsP 3 -binding pocket and channel pore are highly conserved between InsP 3 R family members, the intervening sequence between the binding region and pore is more divergent and consists of the so-called "regulatory and coupling" or "modulatory" domain.…”

mentioning

confidence: 99%

Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

Wagner

Joseph

et al. 2004

Regulation of Ca 2؉ release through inositol 1,4,5-trisphosphate receptors (InsP 3 R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca 2؉ signals. In this study, regulation of Ca 2؉ release by phosphorylation of type 1 InsP 3 R (InsP 3 R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2؉ and S2؊ InsP 3 R-1 splice variants. Ca 2؉ release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP 3 R. In cells expressing either the S1755E S2؉ or S1589E/S1755E S2؊ InsP 3 R-1, InsP 3 -induced Ca 2؉ release was markedly enhanced compared with nonphosphorylatable S2؉ S1755A and S2؊ S1589A/ S1755A mutants. Ca 2؉ release through the S2؊ S1589E/ S1755E InsP 3 R-1 was enhanced ϳ8-fold over wild type and ϳ50-fold when compared with the nonphosphorylatable S2؊ S1589A/S1755A mutant. In cells expressing S2؊ InsP 3 R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca 2؉ release was enhanced ϳ3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/ Glu mutation failed to further enhance Ca 2؉ release. The sensitivity of Ca 2؉ release in cells expressing S2؉ S1755E InsP 3 R-1 was comparable with the sensitivity of S2؊ S1589E/S1755E InsP 3 R-1. In contrast, mutation of S2؉ S1589E InsP 3 R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2؊ S1589E/S1755E InsP 3 R-1 resulted in robust Ca 2؉ oscillations when cells were stimulated with concentrations of ␣-IgM antibody that were threshold for stimulation in S2؊ wild type InsP 3 R-1-expressing cells. However, at higher concentrations of ␣-IgM antibody, Ca 2؉ oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2؊ phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP 3 R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.Inositol 1,4,5-trisphosphate receptors are intracellular ion channels that function to couple the activation of cell surface receptors for neurotransmitters, hormones, and growth factors to the initiation of intracellular Ca 2ϩ release (1). Three genes have been cloned that encode distinct proteins of a molecular mass of ϳ300 kDa, named the type 1 (InsP 3 R-1), 1 type 2 (InsP 3 R-2), and type 3 (InsP 3 R-3) InsP 3 Rs (2-4). In addition, multiple receptor proteins with distinct tissue distributions are produced by alternate splicing of the type 1 receptor gene (5, 6). Most cells express multiple isoforms of InsP 3 R (7). Furthermore, the expression level and complement of receptors differ in individual tissues, and this together with regulation of the activity of the channel is thought to be a major determinant of the rich diversity of Ca 2ϩ signaling events observed ...