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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.

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Structures equivalent to the 4,5-bisphosphate of IP3 are not essential for AdA activity.(A, B) Concentration-dependent effects on Ca2+ release via each IP3R subtype of 4″-dephospho AdA (A) and 3″-dephospho AdA (B) compared with AdA. Results are means ± SEM from n independent experiments (n is provided in Table 1). (C) Concentration-dependent effects of IP3 alone on Ca2+ release via IP3R1 or after pre-incubation (30 s) with 3″-dephospho AdA (30 µM), which itself evoked release of 21±5% of the intracellular Ca2+ stores. Results (C) are means ± SEM from 3 independent experiments.
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pone-0058027-g006: Structures equivalent to the 4,5-bisphosphate of IP3 are not essential for AdA activity.(A, B) Concentration-dependent effects on Ca2+ release via each IP3R subtype of 4″-dephospho AdA (A) and 3″-dephospho AdA (B) compared with AdA. Results are means ± SEM from n independent experiments (n is provided in Table 1). (C) Concentration-dependent effects of IP3 alone on Ca2+ release via IP3R1 or after pre-incubation (30 s) with 3″-dephospho AdA (30 µM), which itself evoked release of 21±5% of the intracellular Ca2+ stores. Results (C) are means ± SEM from 3 independent experiments.

Mentions: 4″-dephospho-AdA at concentrations up to 300 µM failed to evoke Ca2+ release via any IP3R subtype (Figure 6A). This is consistent with previous analyses by both functional and binding assays of IP3R1 [28], [46]. 3″-dephospho-AdA did, however, cause detectable Ca2+ release albeit with much reduced potency (Figure 6B). The synthetic route used to prepare 3″-dephospho-AdA makes it extremely unlikely that the activity could be due to minor contamination with AdA or related structures with a vicinal bisphosphate moiety. Maximal attainable concentrations of 3″-dephospho-AdA (300 µM) failed to release the entire IP3-sensitive Ca2+ store, but comparison of the concentrations required to achieve the same submaximal Ca2+ release suggests that 3″-dephospho-AdA is ∼10,000-fold less potent than AdA at all three IP3R subtypes. With such a massive reduction in potency the lesser sensitivity of DT40-IP3R3 cells to AdA means that even the highest practicable concentration of 3″-dephospho-AdA (300 µM) is close to the threshold for detecting Ca2+ release (Figure 6B).


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 equivalent to the 4,5-bisphosphate of IP3 are not essential for AdA activity.(A, B) Concentration-dependent effects on Ca2+ release via each IP3R subtype of 4″-dephospho AdA (A) and 3″-dephospho AdA (B) compared with AdA. Results are means ± SEM from n independent experiments (n is provided in Table 1). (C) Concentration-dependent effects of IP3 alone on Ca2+ release via IP3R1 or after pre-incubation (30 s) with 3″-dephospho AdA (30 µM), which itself evoked release of 21±5% of the intracellular Ca2+ stores. Results (C) are means ± SEM from 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585173&req=5

pone-0058027-g006: Structures equivalent to the 4,5-bisphosphate of IP3 are not essential for AdA activity.(A, B) Concentration-dependent effects on Ca2+ release via each IP3R subtype of 4″-dephospho AdA (A) and 3″-dephospho AdA (B) compared with AdA. Results are means ± SEM from n independent experiments (n is provided in Table 1). (C) Concentration-dependent effects of IP3 alone on Ca2+ release via IP3R1 or after pre-incubation (30 s) with 3″-dephospho AdA (30 µM), which itself evoked release of 21±5% of the intracellular Ca2+ stores. Results (C) are means ± SEM from 3 independent experiments.
Mentions: 4″-dephospho-AdA at concentrations up to 300 µM failed to evoke Ca2+ release via any IP3R subtype (Figure 6A). This is consistent with previous analyses by both functional and binding assays of IP3R1 [28], [46]. 3″-dephospho-AdA did, however, cause detectable Ca2+ release albeit with much reduced potency (Figure 6B). The synthetic route used to prepare 3″-dephospho-AdA makes it extremely unlikely that the activity could be due to minor contamination with AdA or related structures with a vicinal bisphosphate moiety. Maximal attainable concentrations of 3″-dephospho-AdA (300 µM) failed to release the entire IP3-sensitive Ca2+ store, but comparison of the concentrations required to achieve the same submaximal Ca2+ release suggests that 3″-dephospho-AdA is ∼10,000-fold less potent than AdA at all three IP3R subtypes. With such a massive reduction in potency the lesser sensitivity of DT40-IP3R3 cells to AdA means that even the highest practicable concentration of 3″-dephospho-AdA (300 µM) is close to the threshold for detecting Ca2+ release (Figure 6B).

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
Related in: MedlinePlus