Limits...
Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.

Saleem H, Tovey SC, Riley AM, Potter BV, Taylor CW - PLoS ONE (2013)

Bottom Line: The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3.These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes.They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

Show MeSH
Structures of the analogues of AdA used.(A) Key moieties within IP3 and AdA are highlighted in matching colours to indicate their proposed structural equivalence. (B and C). The Ca2+ contents of the intracellular stores of populations of permeabilized DT40-IP3R1 cells are shown after addition of ATP to allow active Ca2+ uptake, and then addition of the indicated concentrations of IP3 or AdA with thapsigargin (1 µM) to inhibit further Ca2+ uptake. The traces, which are typical of those from all subsequent analyses, show the average response from 2 wells on a single plate. The results demonstrate that both IP3 and AdA evoke quantal Ca2+ release.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585173&req=5

pone-0058027-g001: Structures of the analogues of AdA used.(A) Key moieties within IP3 and AdA are highlighted in matching colours to indicate their proposed structural equivalence. (B and C). The Ca2+ contents of the intracellular stores of populations of permeabilized DT40-IP3R1 cells are shown after addition of ATP to allow active Ca2+ uptake, and then addition of the indicated concentrations of IP3 or AdA with thapsigargin (1 µM) to inhibit further Ca2+ uptake. The traces, which are typical of those from all subsequent analyses, show the average response from 2 wells on a single plate. The results demonstrate that both IP3 and AdA evoke quantal Ca2+ release.

Mentions: Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels that are expressed in almost all animal cells. They allow release of Ca2+ from intracellular stores in response to the many stimuli that activate phospholipase C [1], [2]. The genomes of vertebrates encode three closely related IP3R subtypes (IP3R1-3), and most cells from vertebrates express functional IP3R that are homo- or hetero-tetrameric assemblies of these IP3R subtypes and their splice variants [3]. The physiological significance of this IP3R diversity is poorly understood, and nor are there ligands that usefully discriminate between IP3R subtypes. It is, however, clear that activation of IP3R is initiated by binding of IP3 to the conserved IP3-binding core (IBC, residues 224-604 of IP3R1) of each IP3R subunit [4]. Mixed populations of IP3R in native cells make it difficult to define unambiguously the functional properties of each IP3R subtype. Stable heterologous expression of mammalian IP3R in the only vertebrate cell line engineered to lack all endogenous IP3R (DT40 KO cells) [5] provides an effective means of addressing this difficulty [6]. We previously used DT40 cells expressing homogeneous populations of each mammalian IP3R subtype to define structure-activity relationships for key endogenous and synthetic inositol phosphates [7]. Here, we extend the approach to examine the interactions of each IP3R subtype with adenophostin A (1, AdA) and its most important analogues [8] (Figure 1A).


Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.

Saleem H, Tovey SC, Riley AM, Potter BV, Taylor CW - PLoS ONE (2013)

Structures of the analogues of AdA used.(A) Key moieties within IP3 and AdA are highlighted in matching colours to indicate their proposed structural equivalence. (B and C). The Ca2+ contents of the intracellular stores of populations of permeabilized DT40-IP3R1 cells are shown after addition of ATP to allow active Ca2+ uptake, and then addition of the indicated concentrations of IP3 or AdA with thapsigargin (1 µM) to inhibit further Ca2+ uptake. The traces, which are typical of those from all subsequent analyses, show the average response from 2 wells on a single plate. The results demonstrate that both IP3 and AdA evoke quantal Ca2+ release.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585173&req=5

pone-0058027-g001: Structures of the analogues of AdA used.(A) Key moieties within IP3 and AdA are highlighted in matching colours to indicate their proposed structural equivalence. (B and C). The Ca2+ contents of the intracellular stores of populations of permeabilized DT40-IP3R1 cells are shown after addition of ATP to allow active Ca2+ uptake, and then addition of the indicated concentrations of IP3 or AdA with thapsigargin (1 µM) to inhibit further Ca2+ uptake. The traces, which are typical of those from all subsequent analyses, show the average response from 2 wells on a single plate. The results demonstrate that both IP3 and AdA evoke quantal Ca2+ release.
Mentions: Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels that are expressed in almost all animal cells. They allow release of Ca2+ from intracellular stores in response to the many stimuli that activate phospholipase C [1], [2]. The genomes of vertebrates encode three closely related IP3R subtypes (IP3R1-3), and most cells from vertebrates express functional IP3R that are homo- or hetero-tetrameric assemblies of these IP3R subtypes and their splice variants [3]. The physiological significance of this IP3R diversity is poorly understood, and nor are there ligands that usefully discriminate between IP3R subtypes. It is, however, clear that activation of IP3R is initiated by binding of IP3 to the conserved IP3-binding core (IBC, residues 224-604 of IP3R1) of each IP3R subunit [4]. Mixed populations of IP3R in native cells make it difficult to define unambiguously the functional properties of each IP3R subtype. Stable heterologous expression of mammalian IP3R in the only vertebrate cell line engineered to lack all endogenous IP3R (DT40 KO cells) [5] provides an effective means of addressing this difficulty [6]. We previously used DT40 cells expressing homogeneous populations of each mammalian IP3R subtype to define structure-activity relationships for key endogenous and synthetic inositol phosphates [7]. Here, we extend the approach to examine the interactions of each IP3R subtype with adenophostin A (1, AdA) and its most important analogues [8] (Figure 1A).

Bottom Line: The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3.These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes.They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

Show MeSH