scholarly journals Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization

1999 ◽  
Vol 344 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Jane E. SWATTON ◽  
Stephen A. MORRIS ◽  
Thomas J. A. CARDY ◽  
Colin W. TAYLOR
Keyword(s):  
Single Type ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Rinm5f Cells ◽  
Intact Cells ◽  
Single Receptor ◽  
Insp3 Receptors ◽  
Type 3 ◽  
Insp3 Receptor

There are three subtypes of mammalian Ins(1,4,5)P3 (InsP3) receptor, each of which forms an intracellular Ca2+ channel. Biphasic regulation of InsP3 receptors by cytosolic Ca2+ is well documented in cells expressing predominantly type 1 or type 2 InsP3 receptors and might contribute to the regenerative recruitment of Ca2+ release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP3 receptors of RIN-5F cells, cells in which the InsP3 receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca2+ [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP3 receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP3 receptors in RINm5F cells. In unidirectional 45Ca2+ efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP3 released only a fraction of the InsP3-sensitive Ca2+ stores, indicating that responses to InsP3 are quantal. Increasing the cytosolic free [Ca2+] ([Ca2+]i) from approx. 4 to 186 nM increased the sensitivity of the Ca2+ stores to InsP3: the EC50 decreased from 281±15 to 82±2 nM. Further increases in [Ca2+]i massively decreased the sensitivity of the stores to InsP3, by almost 10-fold when [Ca2+]i was 2.4 μM, and by more than 3000-fold when it was 100 μM. The inhibition caused by 100 μM Ca2+ was fully reversed within 60 s of the restoration of [Ca2+]i to 186 nM. The effect of submaximal InsP3 concentrations on Ca2+ mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca2+. We conclude that type 3 InsP3 receptors of RINm5F cells mediate quantal Ca2+ release and they are biphasically regulated by cytosolic Ca2+, either because a single type 1 subunit within the tetrameric receptor confers the Ca2+ inhibition or because the type 3 subtype is itself directly inhibited by Ca2+.

scholarly journals Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+

1997 ◽  
Vol 328 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Thomas J. A. CARDY ◽  
David TRAYNOR ◽  
W. Colin TAYLOR
Keyword(s):  
Inositol Trisphosphate ◽  
Receptor Subtype ◽  
Sf9 Cells ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Single Mechanism ◽  
Receptor Isoforms ◽  
Type 3

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.

scholarly journals Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors

2000 ◽  
Vol 345 (2) ◽  
pp. 357-363 ◽  
Author(s):  
Charles E. ADKINS ◽  
Stephen A. MORRIS ◽  
Humbert DE SMEDT ◽  
Ilse SIENAERT ◽  
Katalin TÖRÖK ◽  
...  
Keyword(s):  
Calcineurin Inhibitors ◽  
Cell Types ◽  
Receptor Subtypes ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Insp3 Receptors ◽  
Adenophostin A ◽  
A Site ◽  
Insp3 Receptor

InsP3 binding to type-1, but not type-3, InsP3 receptors is inhibited by calmodulin in a Ca2+-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca2+ mobilization by type-1 InsP3 receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP3 receptors, we show that InsP3-evoked Ca2+ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC50 ≈ 15 μM) inhibited InsP3-evoked Ca2+ release only in the presence of Ca2+. The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP3 because calmodulin also decreased the sensitivity of the Ca2+ stores to adenophostin A, a non-metabolizable InsP3-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP3 receptors was unaffected by Ca2+-calmodulin. Using a scintillation proximity assay to measure 125I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP3 receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound 125I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca2+-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP3 receptor inhibits InsP3 binding and may thereby regulate the kinetics of Ca2+ release. Ca2+-dependent inhibition of Ca2+ release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca2+-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca2+ release from any tetrameric receptor that includes a type-1 subunit.

scholarly journals Activation and Deactivation Kinetics of α2A- and α2C-Adrenergic Receptor-activated G Protein-activated Inwardly Rectifying K+Channel Currents

2001 ◽  
Vol 276 (50) ◽  
pp. 47512-47517 ◽  
Author(s):  
Moritz Bünemann ◽  
Markus M. Bücheler ◽  
Melanie Philipp ◽  
Martin J. Lohse ◽  
Lutz Hein
Keyword(s):  
Action Potential ◽  
G Protein ◽  
Adrenergic Receptor ◽  
Hek293 Cells ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Intact Cells ◽  
Deactivation Kinetics ◽  
Inwardly Rectifying ◽  
Kinetics Of

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from α2A-adrenergic receptor knockout mice suggested that the α2A-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the α2C-adrenergic receptor. Here we investigated whether these functional differences between presynaptic α2-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. α2A- and α2C-receptors were stably expressed in HEK293 cells at moderate (∼2 pmol/mg) or high (17–24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K+(GIRK) channels was similar in extent and kinetics for α2A- and α2C-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. α2C-Receptor-activated GIRK currents returned much more slowly to base line than did α2A-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine α2C- than at the α2A-receptor subtype and may explain why α2C-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the α2A-receptor subtype.

Aliskiren, exendin-4, and insulin: their impact on endothelin receptor subtype(s) regulation/binding in type 1 diabetic rat hearts

2013 ◽  
Vol 91 (10) ◽  
pp. 830-838 ◽  
Author(s):  
Sawsan M. Al Lafi ◽  
Shushan B. Artinian ◽  
Suzan S. Boutary ◽  
Nadine S. Zwainy ◽  
Khalil M. Bitar ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Diabetic Rat ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Rat Hearts ◽  
Heart Perfusion ◽  
Glucagon Like Peptide 1 ◽  
Coronary Endothelium ◽  
The Impact

This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/regulation of endothelin-1 (ET-1) to its receptor subtypes A (ETAR) and B (ETBR) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [125I]-ET-1 was employed to estimate ET-1 binding affinity (τ = 1/K–n) to ETAR and ETBR at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of ETAR and ETBR were detected using Western blot. No significance differences were detected in the τ of ETAR and ETBR between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the τ value for ETAR and ETBR on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, ETAR and ETBR densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated ETAR and down-regulated ETBR band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus.

scholarly journals Human Types 1 and 3 3α-Hydroxysteroid Dehydrogenases: Differential Lability and Tissue Distribution1

2001 ◽  
Vol 86 (2) ◽  
pp. 841-846 ◽  
Author(s):  
Isabelle Dufort ◽  
Fernand Labrie ◽  
Van Luu-The
Keyword(s):  
Hacat Keratinocytes ◽  
Human Embryonic Kidney ◽  
Intact Cells ◽  
Hek 293 ◽  
Human Type ◽  
Hydroxysteroid Dehydrogenases ◽  
293 Cells ◽  
Hydroxy Compounds ◽  
Type 3

3α-Hydroxysteroid dehydrogenases (3α-HSDs) catalyze the conversion of 3-ketosteroids to 3α-hydroxy compounds. The best known 3α-HSD activity is the transformation of the most potent natural androgen, dihydrotestosterone, into 5α-androstan-3α,17β-diol (3α-diol), a compound having much lower activity. Previous reports show that 3α-HSDs are involved in the metabolism of glucocorticoids, progestins, prostaglandins, bile acid precursors, and xenobiotics. 3α-HSDs could, thus, play a crucial role in the control of a series of active steroid levels in target tissues. In the human, type 1 3α-HSD was first identified as human chlordecone reductase. Recently, we have isolated and characterized type 3 3α-HSD that shares 81.7% identity with human type 1 3α-HSD. The transfection of vectors expressing types 1 and 3 3α-HSD in transformed human embryonic kidney (HEK-293) cells indicates that both enzymes efficiently catalyze the transformation of dihydrotestosterone into 3α-diol in intact cells. However, when the cells are broken, the activity of type 3 3α-HSD is rapidly lost, whereas the type 1 3α-HSD activity remains stable. We have previously found that human type 5 17β-HSD which possesses 84% and 86% identity with types 1 and 3 3α-HSD, respectively, is also labile, whereas rodent enzymes such as mouse type 5 17β-HSD and rat 3α-HSD are stable after homogenization of the cells. The variable stability of different enzymatic activities in broken cell preparations renders the comparison of different enzymes difficult. RNA expression analysis indicates that human type 1 3α-HSD is expressed exclusively in the liver, whereas type 3 is more widely expressed and is found in the liver, adrenal, testis, brain, prostate, and HaCaT keratinocytes. Based on enzymatic characteristics and sequence homology, it is suggested that type 1 3α-HSD is an ortholog of rat 3α-HSD while type 3 3α-HSD, which must have diverged recently, seems unique to human and is probably more involved in intracrine activity.

scholarly journals Regulation by Ca2+ and Inositol 1,4,5-Trisphosphate (Insp3) of Single Recombinant Type 3 Insp3 Receptor Channels

2001 ◽  
Vol 117 (5) ◽  
pp. 435-446 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Sean McBride ◽  
J. Kevin Foskett
Keyword(s):  
Mammalian Cells ◽  
Single Channel ◽  
Channel Gating ◽  
Hill Coefficient ◽  
Release Channel ◽  
Recombinant Type ◽  
Inositol 1,4,5 Trisphosphate ◽  
Type 3 ◽  
Insp3 Receptor

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is an endoplasmic reticulum–localized Ca2+-release channel that controls complex cytoplasmic Ca2+ signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 InsP3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of ∼3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 μM under saturating (10 μM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP3 concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of ∼4. InsP3 activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3–induced Ca2+ release and low gain Ca2+–induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.

scholarly journals Simple method for distinguishing gonococcal colony types

1977 ◽  
Vol 6 (5) ◽  
pp. 511-517
Author(s):  
E Juni ◽  
G A Heym
Keyword(s):  
Incident Light ◽  
Rapid Identification ◽  
Dark Field ◽  
Single Type ◽  
Strain Identification ◽  
Concave Mirror ◽  
Simple Method ◽  
Focusing Effect ◽  
Type 3

Gonococcal colony types can be distinguished by a new procedure that makes use of a dissecting microscope with a concave mirror and a fluorescent lamp. Critical adjustment of the mirror angle results in illumination similar to that obtained in the dark-field microscope. When the concave mirror is set at a certain angle, colonies of the lenticular types 1 and 2 refract the light coming through them in such a way that an edge of the microscope stage is focused in each colony. By contrast, colonies of types 3 and 4, which are relatively flat, fail to refract incident light. Although distinguishable from each other by differences in color, type 3 and 4 colonies do not display the focusing effect typical for type 1 and 2 colonies and appear uniformly illuminated. This new technique permits the rapid identification and isolation of even a single type 1 or 2 colony in a field of type 3 or 4 colonies, making it possible to obtain and maintain competent colonies (type 1 or 2) for the genetic transformation assay for Neisseria gonorrhoeae strain identification as well as for other purposes.

scholarly journals Expression of type 1 angiotensin II receptor subtypes and angiotensin II-induced calcium mobilization along the rat nephron.

1997 ◽  
Vol 8 (11) ◽  
pp. 1658-1667 ◽  
Author(s):  
N Bouby ◽  
A Hus-Citharel ◽  
J Marchetti ◽  
L Bankir ◽  
P Corvol ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Collecting Duct ◽  
At1 Receptor ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Angiotensin Ii Receptor ◽  
Nephron Segments

The localization of two type 1 angiotensin II receptor subtype mRNA, AT1A and AT1B, was determined by reverse transcription-PCR on microdissected glomeruli and nephron segments. The coupling sensitivity of these two receptor subtypes was evaluated by measuring variations in intracellular calcium ([Ca2+]i) elicited by angiotensin II (Ang II) in structures expressing either AT1A or AT1B mRNA, using Fura-2 fluorescence. The highest expression of AT1 mRNA was found in glomerulus, proximal tubule, and thick ascending limb. In glomerulus, AT1A and AT1B mRNA were similarly expressed, whereas in all nephron segments AT1A mRNA expression was dominant (approximately 84%). The increase in [Ca2+]i elicited by 10(-7) mol/L Ang II was highest in proximal segments (delta [Ca2+]i is approximately equivalent to 300 to 400 nmol/L) and thick ascending limb (delta [Ca2+]i is approximately equivalent to 200 nmol/L). In glomerulus and collecting duct, the response was lower (delta < 100 nmol/L). The median effective concentrations for Ang II were of the same order of magnitude in glomerulus (12.2 nmol/L), in which both AT1A and AT1B are expressed, and in cortical thick ascending limb (10.3 nmol/ L), in which AT1A is almost exclusively expressed. The Ang II-induced calcium responses were totally abolished by the AT1 receptor antagonist losartan (1 mumol/L) but not by the AT2 antagonist PD 123319 (1 mumol/L). In the absence of external Ca2+, the peak phase of the response induced by 10(-7) mol/L Ang II was reduced and shortened, suggesting that a part of the [Ca2+]i increase originated from the mobilization of the intracellular Ca2+ pool. In conclusion, these results demonstrate that in the rat kidney: (1) AT1A is the predominant AT1 receptor subtype expressed in the nephron segments, (2) glomerulus is the only structure with a relatively high AT1B mRNA content, and (3) AT1A and AT1B receptor subtypes do not differ in their efficiency for the activation of calcium second-messenger system.

Agonist selectivity in the oxytocin/vasopressin receptor family: new insights and challenges

2007 ◽  
Vol 35 (4) ◽  
pp. 737-741 ◽  
Author(s):  
B. Chini ◽  
M. Manning
Keyword(s):  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Bivalent Ligands ◽  
Single Receptor ◽  
Vasopressin Receptors ◽  
Selective Agonists ◽  
Receptor Assays ◽  
Definition Of ◽  
High Degree ◽  
Subtype Selective

The design and development of selective agonists acting at the OT (oxytocin)/AVP (vasopressin) receptors has been and continues to be a difficult task because of the great similarity among the different receptor subtypes as well as the high degree of chemical similarity between the active ligands. In recent decades, at least a thousand synthetic peptides have been synthesized and examined for their ability to bind to and activate the different OT/AVP receptors; an effort that has led to the identification of several receptor subtype-selective agonists in the rat. However, owing to species differences between rat and human AVP/OT receptors, these peptides do not exhibit the same selectivities in human receptor assays. Furthermore, the discovery of receptor promiscuity, which is the ability of a single receptor subtype to couple to several different G-proteins, has led to the definition of a completely new class of compounds, referred to here as coupling-selective ligands, which may activate, within a single receptor subtype, only a specific signalling pathway. Finally, the accumulating evidence that GPCRs (G-protein-coupled receptors) do not function as monomers, but as dimers/oligomers, opens up the design of another class of specific ligands, bivalent ligands, in which two agonist and/or antagonist moieties are joined by a spacer of the appropriate length to allow the simultaneous binding at the two subunits within the dimer. The pharmacological properties and selectivity profiles of these bivalent ligands, which remain to be investigated, could lead to highly novel research tools and potential therapeutic agents.

Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor

2001 ◽  
Vol 355 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Edmund P. NEROU ◽  
Andrew M. RILEY ◽  
Barry V. L. POTTER ◽  
Colin W. TAYLOR
Keyword(s):  
Hydroxy Group ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Subtype Selectivity ◽  
Type 3

Synthetic analogues of inositol trisphosphate (IP3), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP3, were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP3 receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [3H](1,4,5)IP3 in Ca2+-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP3 were type 3 (Kd = 11±2nM)>type 2 (Kd = 17±2nM) > type 1 (Kd = 24±4nM). (1,4,5)IP3 binding was reversibly stimulated by increased pH, but the subtypes differed in their sensitivity to pH (type 1 > type 2>type 3). For all three subtypes, the equatorial 6-hydroxy group of (1,4,5)IP3 was essential for high-affinity binding, the equatorial 3-hydroxy group significantly improved affinity, and the axial 2-hydroxy group was insignificant; a 1-phosphate (or in its absence, a 2-phosphate) improved binding affinity. The subtypes differed in the extents to which they tolerated inversion of the 3-hydroxy group of (1,4,5)IP3 (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP3 for type 1 receptors. They also differed in their tolerance of inversion, removal or substitution (by phosphate) of the 2-hydroxy group (types 2 and 3>type 1), hence the selectivity of (1,2,4,5)IP4 for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH2OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP3 for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP3 receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP3 as key determinants of subtype selectivity.

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