Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium

Abstract

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.

Defining the minimal structural requirements for partial agonism at the type I myo‐inositol 1,4,5‐trisphosphate receptor

“…Whilst Ins(l,4,5)PS 3 has only a slightly lower affinity than Ins(l,4,5)P 3 at binding sites characterised with [ 3 H]Ins(l,4,5)P 3 , inositol l,4,5-[ 35 S(U)]trisphosphorothioate was able to specifically label two specific binding site populations [6]. Thus,Ins(l,4,5)PS 3 appears to exhibit a subtly different interaction with the binding pocket of the Ins(l,4,5)P 3 R [Ins(l,4,5)P 3 R].…”

“…Statistical comparison of the logio[IC5o] and logio[EC5o] was performed in Excel (Microsoft, UK), using unpaired Student's Mests (assuming unequal variances) at the p £ 0.05 level. ( 1,4,5 ) Fig. 2).…”

“…The interaction of Ins(l,4,5)P 3 with its receptor is highly stereospecific, the vicinal 4,5-bisphosphate and 6-hydroxyl motif being the crucial structural features, and the 1-phosphate of lns (l,4,5)P3 further contributing to receptor binding specificity (reviewed [2]). Partial or total phosphorothioate substitution of the phosphate groups appears to well tolerated, producing high affinity ligands which are resistant to metabolism catalysed by the specific enzymes Ins(l,4,5)P 3 -3-kinase and Ins(l,4,5)P 3 -5-phosphatase [3][4][5]. Whilst Ins(l,4,5)PS 3 has only a slightly lower affinity than Ins(l,4,5)P 3 at binding sites characterised with [ 3 H]Ins(l,4,5)P 3 , inositol l,4,5-[ 35 S(U)]trisphosphorothioate was able to specifically label two specific binding site populations [6].…”

“…In contrast, inversion of the 3-OH [12,13] produces loss of activity, while increasing the steric bulk of the 3-position substituent of Ins(l,4,5)P 3 analogues correlates with progressively decreasing activity at the Ins(l,4,5)P 3 R [14]. Notably some modifications of the equatorial 3-position hydroxyl of Ins(l,4,5)P 3 affect the intrinsic activity at the Ins(l,4,5)P 3 R. Indeed, the first partial agonist identified at the Ins(l,4,5)P 3 R was the naturally occurring myo-inositol 1,3,4,3,4,6)P 4 ] [11,15]; significantly Ins (l,3,4,6)P4 adopts a binding conformation which efficiently mimics Ins(l,4,5)P 3 with the exception of presenting an axial (pseudo) 3-phosphate [7]. Additionally, recently two low intrinsic activity partial agonists; h-chiroinositol 2,3,3,5)PS 3 ], D- 4,4,5)PS 3 ] [16] and a high intrinsic activity partial agonist; scy/fo-inositol 1,2,4,5-tetrakisphosphorothioate [11] have been identified.…”

“…D-wyo- Inositol 1,4,4,5)P 3 ] receptors are recognised as a homologous family of tetrameric ligandgated Ca 2+ channels, which allow mobilisation of intracellular Ca 2+ stores in response to activation of cell-surface receptors linked to Ins(l,4,5)P 3 generation [1]. The interaction of Ins(l,4,5)P 3 with its receptor is highly stereospecific, the vicinal 4,5-bisphosphate and 6-hydroxyl motif being the crucial structural features, and the 1-phosphate of lns (l,4,5)P3 further contributing to receptor binding specificity (reviewed [2]).…”