Location of the permeation pathway in the recombinant type 1 inositol 1, 4, 5-trisphosphate receptor (original) (raw)
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120 Transmembrane topology and sites of n-glycosylation of inositol 1,4,5-trisphosphate receptor
Neuroscience Research Supplements, 1993
To define the transmembrane topology of the inositol 1,4,5-trisphosphate receptor (InsP3R), we determined the subcellular location of the hydrophilic segment (residues 2463-2529 of mouse type 1 InsF3R) believed to be located at the luminal side of the endoplasmic reticulum (ER) in the six-transmembrane model but at the cytoplasmic side in the eight-transmembrane model.
The Journal of General Physiology, 2012
generated in the cytoplasm in response to extracellular stimuli binds to and activates InsP 3 R channels to release Ca 2+ stored in Correspondence to Don-On Daniel Mak: d m a k @ m a i l . m e d . u p e n n . e d u Abbreviations used in this paper: HEDTA, hydroxyethylethylenediaminetriacetic acid; InsP 3 , inositol 1,4,5-trisphosphate; InsP 3 R, InsP 3 receptor; lum-out, luminal-side-out; P o , open probability; r-InsP 3 R-3, rat type 3 InsP 3 R. the ER lumen into the cytoplasm, generating diverse local and global [Ca 2+ ] i signals . Whereas much is known regarding the intricate regulation of InsP 3 R channel gating by multiple processes-binding of cytoplasmic ligands (Ca 2+ , InsP 3 , and ATP 4 ), posttranslational modifications, interactions with proteins, clustering, dif-The ubiquitous inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) Ca 2+ release channel plays a central role in the generation and modulation of intracellular Ca 2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP 3 , free Ca 2+ , free ATP 4 ) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP 3 R channel activity by free Ca 2+ in the ER lumen ([Ca 2+ ] ER ) remains poorly understood because of limitations of Ca 2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca 2+ ] ER on homotetrameric rat type 3 InsP 3 R channel activity. In optimal [Ca 2+ ] i and subsaturating [InsP 3 ], jumps of [Ca 2+ ] ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP 3 but restored when [Ca 2+ ] ER was raised to 1.1 mM. In suboptimal [Ca 2+ ] i , jumps of [Ca 2+ ] ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca 2+ ] ER effects on channel activity exhibited a biphasic dependence on [Ca 2+ ] i . In addition, the effect of high [Ca 2+ ] ER was attenuated when a voltage was applied to oppose Ca 2+ flux through the channel. These observations can be accounted for by Ca 2+ flux driven through the open InsP 3 R channel by [Ca 2+ ] ER , raising local [Ca 2+
The Journal of General Physiology, 1998
The inositol (1,4,5)-trisphosphate receptor (InsP3R) mediates Ca2+release from intracellular stores in response to generation of second messenger InsP3. InsP3R was biochemically purified and cloned, and functional properties of native InsP3-gated Ca2+channels were extensively studied. However, further studies of InsP3R are obstructed by the lack of a convenient functional assay of expressed InsP3R activity. To establish a functional assay of recombinant InsP3R activity, transient heterologous expression of neuronal rat InsP3R cDNA (InsP3R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP3R retained specific InsP3binding properties (Kd= 60 nM InsP3) and were specifically recognized by anti–InsP3R-I rabbit polyclonal antibody. Density of expressed InsP3R-I was at least 20-fold above endogenous InsP3R background and only 2–3-fold lower than InsP3R density in rat cerebellar microsomes. When incorporated i...
2001
We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca 2؉ release channel (inositol 1,4,5-trisphosphate receptors (IP 3 Rs)) that is gated by the second messenger inositol 1,4,5trisphophate (IP 3). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a 45 Ca 2؉ flux assay and single channel electrophysiological analyses. Two IP 3 R mutations, V2548I and D2550E, retained the ability to release 45 Ca 2؉ in response to IP 3. When analyzed at the single channel level; both recombinant channels had IP 3-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K ؉ conductance (489 ؎ 13 picosiemens (pS) for V2548I versus 364 ؎ 5 pS for wildtype), but retained a Ca 2؉ selectivity similar to wild-type channels (P Ca 2؉:P K ؉ ϳ 4:1). Conversely, D2550E channels were nonselective for Ca 2؉ over K ؉ (P Ca 2؉:P K ؉ ϳ 0.6:1), while the K ؉ conductance was effectively unchanged (391 ؎ 4 pS). These results suggest that amino acid residues Val 2548 and Asp 2550 contribute to the ion conduction pathway. We propose that the pore of IP 3 R channels has two distinct sites that control monovalent cation permeation (Val 2548) and Ca 2؉ selectivity (Asp 2550). In response to a wide variety of external stimuli, the second messenger inositol 1,4,5-trisphosphate (IP 3) 1 is generated from * This work was supported by National Institutes of Health Grants R01-DK34804 (to S. K. J.) and R01-MH59937 (to J. K. F.), a predoctoral fellowship from training Grant T32-AA07463 (to D. B.) from the National Institutes of Health, and by American Heart Association Grant 9906220U (D.-O. D. M.
Carboxyl-terminal sequences critical for inositol 1,4,5-trisphosphate receptor subunit assembly
The Journal of biological chemistry, 2002
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 -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 ϳ30 residues in length and is localized between residues 2629 and 2654. These residues are highly conserved between the three InsP 3 R isoforms (ϳ80% identity) as well as the ryanodine receptor (ϳ40% 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 membranespanning regions and residues 2629 -2654 of the carboxyl terminus possibly through the formation of a dimer of dimers.
Journal of Biological Chemistry, 1997
Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) by Ca 2؉ . To define the structural determinants for Ca 2؉ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP 3 R-1 were expressed in bacteria. The fusion proteins were screened for Ca 2؉ and ruthenium red binding through the use of 45 Ca 2؉ and ruthenium red overlay procedures. Six new cytosolic Ca 2؉ -binding regions were detected on the InsP 3 R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012). Strong 45 Ca 2؉ and ruthenium red binding domains were localized in the N-terminal region of the InsP 3 R as follows: two Ca 2؉ -binding domains were located within the InsP 3 -binding domain, and three Ca 2؉ binding stretches were localized in a 500-amino acid region just downstream of the InsP 3 -binding domain. A sixth Ca 2؉ -binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca 2؉ -binding sites in the regulation of the InsP 3 R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca 2؉ and Sr 2؉ on the EC 50 and cooperativity of the InsP 3 -induced Ca 2؉ release. The activation by cytosolic Ca 2؉ was due to a shift in EC 50 toward lower InsP 3 concentrations, and this effect was mimicked by Sr 2؉ . The inhibition by cytosolic Ca 2؉ was caused by a decrease in cooperativity and by a shift in EC 50 toward higher InsP 3 concentrations. The effect on the cooperativity occurred at lower Ca 2؉ concentrations than the inhibitory effect on the EC 50 . In addition, Sr 2؉ mimicked the effect of Ca 2؉ on the cooperativity but not the inhibitory effect on the EC 50 . The different [Ca 2؉ ] and [Sr 2؉ ] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca 2؉ stimulated the release without affecting the Hill coefficient or the EC 50 , excluding the involvement of one of the cytosolic Ca 2؉ -binding sites. We conclude that multiple Ca 2؉ -binding sites are localized on the InsP 3 R-1 and that at least four different Ca 2؉ -interaction sites may be involved in the complex feedback regulation of the release by Ca 2؉ .
Inositol 1,4,5-trisphosphate receptors in the endoplasmic reticulum: A single-channel point of view
Cell calcium, 2014
As an intracellular Ca(2+) release channel at the endoplasmic reticulum membrane, the ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) plays a crucial role in the generation, propagation and regulation of intracellular Ca(2+) signals that regulate numerous physiological and pathophysiological processes. This review provides a concise account of the fundamental single-channel properties of the InsP3R channel: its conductance properties and its regulation by InsP3 and Ca(2+), its physiological ligands, studied using nuclear patch clamp electrophysiology.
Critical Regions for Activation Gating of the Inositol 1,4,5-Trisphosphate Receptor
Journal of Biological Chemistry, 2003
did not exhibit any measurable Ca 2؉ release activity, whereas the mutants lacking residues 1131-1379 and 2736 -2749 retained the activity. Limited trypsin digestion showed that not only the IP 3 -gated Ca 2؉ -permeable mutants lacking residues 1131-1379 and 2736 -2749, but also two nonfunctional mutants lacking residues 1-223 and 651-1130, retained the normal folding structure of at least the C-terminal channel-forming domain. These results indicate that two regions of IP 3 R1, viz. residues 1-223 and 651-1130, are critical for IP 3 -induced gating. We also identified a highly conserved cysteine residue at position 2613, which is located within the C-terminal tail, as being essential for channel opening. Based on these results, we propose a novel five-domain structure model in which both N-terminal and internal coupling domains transduce ligand-binding signals to the C-terminal tail, which acts as a gatekeeper that triggers opening of the activation gate of IP 3 R1 following IP 3 binding.