Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a tetrameric intracellular Ca(2+) channel, which mediates the release of Ca(2+) from the endoplasmic reticulum in response to many different extracellular stimuli. We present a 3D structure of the type 1 IP(3)R obtained by electron microscopy and single-particle analysis that reveals its domain organization. The IP(3)R has a flower-like appearance with fourfold symmetry and is made up of three distinct domains connected by slender links. By relating the organization of the structural domains to secondary-structure predictions and biochemical data we develop a model in which structural domains are mapped onto the amino acid sequence to deduce the location of the channel region and the cytoplasmic inositol 1,4,5-trisphosphate-binding and modulatory subdomains. The structure of the IP(3)R is compared with that of other tetrameric cation channels. The channel domain is similar in size and shape to its counterparts in the ryanodine receptor and the Shaker voltage-gated K(+) channel.

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 (IP(3))-binding sites of tetrameric IP(3) receptors were probed using dimers of IP(3) linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated (45)Ca(2+) release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP(3) 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 IP(3). 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 IP(3). The affinity of monomeric IP(3) with only the PEG attached was 2-fold weaker than IP(3), confirming that the increased affinity of the dimers requires the presence of both IP(3) motifs. The increased affinity of the long dimer probably results from the linked IP(3) molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP(3) to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP(3) to purified receptors. IP(3) and the IP(3) dimers, irrespective of their length, bound with similar affinity to a monomeric IP(3)-binding domain of the type 1 IP(3) receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP(3)-binding sites of an IP(3) receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 x 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP(3)-binding sites of the IP(3) receptor.

Determinants of adenophostin A binding to inositol trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP(3)) receptors from cerebellum and recombinant type 1 IP(3) receptors expressed in Sf9 cells had indistinguishable affinities for IP(3) ( K (d)=6.40+/-0.48 nM) and adenophostin A ( K (d)=0.89+/-0.05 nM). In cytosol-like medium, each of the three mammalian IP(3) receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP(3). It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [(3)H]IP(3) from type 1 IP(3) receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [(3)H]IP(3) binding to the 1-604 protein, but abolished binding to the 224-604 protein. The 1-604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP(3), suggesting that C-terminal residues selectively inhibit IP(3) binding. The 224-604S1(+) fragment bound IP(3) and adenophostin A with increased affinity, but as with the 1-604 fragment it bound adenophostin A with only 2-fold greater affinity than IP(3). High-affinity binding of adenophostin A may be partially determined by its 2'-phosphate interacting more effectively than the 1-phosphate of IP(3) with residues within the IP(3)-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP(3) for the minimal IP(3)-binding domain. In addition we suggest that C-terminal residues, which impede access of IP(3), may selectively interact with adenophostin A to allow it unhindered access to the IP(3)-binding domain.

A novel Ca2+-induced Ca2+ release mechanism mediated by neither inositol trisphosphate nor ryanodine receptors.

Members of both major families of intracellular Ca(2+) channels, ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, are stimulated by substantial increases in cytosolic free Ca(2+) concentration ([Ca(2+)]c). They thereby mediate Ca(2+)-induced Ca(2+) release (CICR), which allows amplification and regenerative propagation of intracellular Ca(2+) signals. In permeabilized hepatocytes, increasing [Ca(2+)]c to 10 microM stimulated release of 30+/-1% of the intracellular stores within 60 s; the EC(50) occurred with a free [Ca(2+)] of 170+/-29 nM. This CICR was abolished at 2 degrees C. The same fraction of the stores was released by CICR before and after depletion of the IP3-sensitive stores, and CICR was not blocked by antagonists of IP3 receptors. Ryanodine, Ruthenium Red and tetracaine affected neither the Ca(2+) content of the stores nor the CICR response. Sr(2+) and Ba(2+) (EC(50)=166 nM and 28 microM respectively) mimicked the effects of increased [Ca(2+)] on the intracellular stores, but Ni(2+) blocked the passive leak of Ca(2+) without blocking CICR. In rapid superfusion experiments, maximal concentrations of IP3 or Ca(2+) stimulated Ca(2+) release within 80 ms. The response to IP3 was complete within 2 s, but CICR continued for tens of seconds despite a slow [half-time (t(1/2))=3.54+/-0.07 s] partial inactivation. CICR reversed rapidly (t(1/2)=529+/-17 ms) and completely when the [Ca(2+)] was reduced. We conclude that hepatocytes express a novel temperature-sensitive, ATP-independent CICR mechanism that is reversibly activated by modest increases in [Ca(2+)], and does not require IP3 or ryanodine receptors or reversal of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase. This mechanism may both regulate the Ca(2+) content of the intracellular stores of unstimulated cells and allow even small intracellular Ca(2+) signals to be amplified by CICR.