143P University of Cambridge
Summer Meeting July 2005

 

Interaction between inositol 1,4,5-trisphosphate (IP3) receptors and dimers of IP3

Ana M. Rossi, Stephen C. Tovey, Emily J. A. Taylor, Skarlatos G. Dedos, Andrew M. Riley 1, Barry V. L. Potter 1 and Colin W. Taylor. Department of Pharmacology, Cambridge and 1Department of Pharmacy and Pharmacology, Bath.

 

IP3 receptors (IP3R) belong to a family of intracellular Ca2+ -release channels, which are expressed largely in the endoplasmic reticulum of almost all cells (Taylor et al., 2002). Three mammalian genes (types 1–3) have been cloned, each encoding a polypeptide of ~2700 residues. IP3R assemble into homo or hetero-tetramers, and each subunit contains a single IP3-binding site towards the N-terminus (Taylor et al., 1999).

Dimers of IP3 linked either directly or by polyethylene glycol spacers of different lengths were synthesized and their effects examined in functional and binding assays. DT40KO cells, in which the genes for all 3 endogenous IP3R subtypes are deleted (Sugawara, et al., 1997), were transfected with pcDNA3-IP3R1to produce cells stably expressing only rat IP3R1. Ca2+ release from intracellular stores was monitored in these permeabilized cells using Mag-Fluo-4AM in a Flexstation fluorescence plate reader. Radioligand binding assays were performed on full-length IP3R1 purified from rat cerebellum and bacterially expressed N-terminal fragments of IP3R1.

Each of the dimers examined (with the IP3 moieties separated by 0.8nm - 8nm), released the same fraction of the intracellular stores as IP3, but each was more potent than IP3. In equilibrium competition binding assays to IP3R1, the rank order of affinity of the dimeric analogues of IP3 matched that obtained from functional studies (Table I). The shortest dimer (separation 0.8nm) was the most potent: it bound with (25 ± 3)-fold greater affinity than IP3 and caused Ca2+ release at (8.1 ± 1.2)-fold lower concentrations. A monomeric N-terminal domain (residues 1-604) of the IP3 receptor likewise bound the dimer with (9.8 ± 1.9)-fold greater affinity than IP3, but the shorter “IP3-binding core” (residues 224-604) bound IP3 and dimeric IP3 with similar affinity (KD = 0.14 ± 0.08 and 0.14 ± 0.02 nM, respectively).

We conclude that the high-affinity of dimeric IP3 requires interactions with both the binding core (shared with IP3) and with residues in the extreme N-terminus of the IP3R.

 

Analogue

Ca2+ release (EC50 nM)

Binding to IP3R1 (KD nM)

IP3

20.5 ± 1.6

10.9 ± 2.87

0.8 nm dimer

2.7 ± 0.4

0.43 ± 0.21

1.5 nm dimer

4.9 ± 0.3

0.79 ± 0.21

8 nm dimer

12.9 ± 0.8

1.29 ± 0.26

 

Taylor , et al. (2002) Cell Calcium. 32, 321-334.
Taylor , et al. (1999) Cell Calcium. 26, 237-251.
Sugawara, et al. (1997) EMBO. J. 16, 3078-3088.

Supported by the Wellcome Trust (072084), the Gates Cambridge Trust and the Overseas Research Trust.