Interactions of inositol 1,4,5-trisphosphate (IP3) receptors with synthetic poly(ethylene glycol)-linked dimers of IP3 suggest close spacing of the IP3-binding sites

Abstract

The distances between the inositol 1,4,5-trisphosphate (IP3)-binding sites of tetrameric IP3 receptors were probed using dimers of IP3 linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated 45Ca2+ 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. IP3 and 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.