Vanessa Correa

Interactions of inositol 1,4,5-trisphosphate (IP(3)) receptors with synthetic poly(ethylene glycol)-linked dimers of IP(3) suggest close spacing of the IP(3)-binding sites.

The distances between the inositol 1,4,5-trisphosphate (IP3)-binding sites of tetrameric IP3 receptors were probed using dimers of IP3linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1–8 nm). Each of the dimers potently stimulated45Ca2+ release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP3 receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3–4-fold more potent than IP3. In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP3. The affinity of monomeric IP3 with only the PEG attached was 2-fold weaker than IP3, confirming that the increased affinity of the dimers requires the presence of both IP3 motifs. The increased affinity of the long dimer probably results from the linked IP3 molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP3 to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP3 to purified receptors. IP3and the IP3 dimers, irrespective of their length, bound with similar affinity to a monomeric IP3-binding domain of the type 1 IP3 receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP3-binding sites of an IP3 receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 × 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP3-binding sites of the IP3 receptor.

Synthesis of adenophostin A

Abstract The natural product and potent agonist of the d - myo -inositol 1,4,5-trisphosphate receptor, adenophostin A, was synthesised from adenosine and d -glucose using efficient methodology. The synthetic material was equipotent with naturally occurring adenophostin A in evoking Ca 2+ release from the intracellular stores of permeabilised cells.

INFRAMOLECULAR STUDIES OF THE PROTONATION OF 1D-1,2,4/3,5-CYCLOPENTANEPENTAOL 1,3,4-TRISPHOSPHATE, A RING-CONTRACTED ANALOGUE OF 1D-MYO-INOSITOL 1, 4, 5-TRISPHOSPHATE

Abstract The protonation process of the individual phosphate groups of 1 d -1,2,4/3,5-cyclopentanepentol 1,3,4-trisphosphate has been investigated by 31P NMR titration experiments. The results are compared with those of 1 d -myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. From the NMR titration curves it can be shown that P3 and P4 strongly interact, whereas P1 behaves independently. In addition, P3 and P4 are much less influenced by their vicinal hydroxyls than P1. Accordingly, the calculated microscopic protonation constants indicate a higher basicity for P3 and P4 compared with the basicity of P1. The vicinal bisphosphate of the cyclopentane-based compounds seems to adopt a similar conformation to that of Ins(1,4,5)P3, although an analogous interaction with a hydroxyl group is not observed. Two cyclopentanepentaol trisphosphates were examined for their potency to release intracellular Ca2+ from rat hepatocytes. Both were found to be low-affinity agonists in this regard. The relationship of this activity to the physicochemical data is discussed for one example.