Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors

Abstract

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca 2؉ -induced conformational changes of IP 3 Rs in their native membrane environment. We found that Ca 2؉ decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca 2؉ induces a conformational change in the IP 3 R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca 2؉ were observed at ϳ0.5 M and was sensitive to inhibition by IP 3 . Ca 2؉ also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1-225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca 2؉ . However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca 2؉ . We conclude the following: a) that large conformational changes induced by Ca 2؉ can be detected in IP 3 Rs in situ; b) these changes may be driven by Ca 2؉ binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP 3 R may be relatively inaccessible to regulatory proteins unless Ca 2؉ is present.Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 3 are large tetrameric intracellular Ca 2ϩ -release channels that mediate the release of Ca 2ϩ from endoplasmic reticulum stores into the cytosol in response to IP 3 (1-5). Three different isoforms exist, which can form homo-and heteroligomers. All three isoforms consist of an IP 3 binding domain in the N-terminal region, a channel domain containing six transmembrane segments in the C-terminal region and an intervening regulatory domain. Apart from IP 3 , the principal regulator of the channel is Ca 2ϩ , which exerts a biphasic effect on channel function with activation at low concentrations and inhibition at high concentrations. The structural organization of this complex protein and the mechanism by which binding of IP 3 and Ca 2ϩ are linked to channel gating are active areas of investigation.An important feature of the mechanism of the channel involves conformational changes in the protein. In particular, the gating mechanism is thought to involve conformational changes initiated by IP 3 binding that are propagated to various regions of the channel domain (6, 7). The IP 3 binding domain of the receptor is organized into a "core" segment that binds IP 3 with high affinity (amino acids 226 -604) and a "suppressor domain" that inhibits IP 3 binding to the core (amino acids 1-225). Crystal structures for the suppressor domain and the ...

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

“…6B. The main assumption of the model, based on previous cysteine accessibility studies (52), is that the C-terminal tail projects all the way to the cytosolic surface within the central portion of the receptor assembly. In this model we suggest that one mechanism by which IP 3 induced conformational changes in the LBD could be transmitted to the channel domain is by altering the conformation of the C-terminal tail.…”

Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis

“…Given the absence of any further known predicted nucleotide binding sites in the primary sequence, a novel ATP binding sequence must mediated these effects. A possibility is that the interaction is dependent on the tertiary structure of the InsP 3 R. Alternatively, a cryptic ATP binding site may be exposed following the large conformational changes the receptor undergoes when binding Ca 2+ [68-70]. Conceivably, the functional effects of ATP could also be mediated through a tightly bound accessory protein.…”

“…However, reaction of cysteine substitution mutants introduced into the C-tail of full length receptors with 5kDa PEG-maleimide suggest that freely accessible residues are limited to the terminal ~35 amino-acids. Using this assay, Ca 2+ -induced conformational changes in the receptor were observed to enhance accessibility of the C-tail[68]. Structural information on the C-tail, particularly its orientation with respect to the membrane and the rest of the receptor, is needed to determine its role in InsP 3 R function and regulation.…”

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

“…Two intrinsic determinants stabilizing the subunit configurations can be considered a priori-IP 3 and Ca 2+ -because channel activation requires both IP 3 and Ca 2+ . In fact, the recent X-ray structures of the IBD indicate that conformational changes occur upon IP 3 binding (7,8); our group and others have demonstrated Ca 2+ -induced structural changes (84,85,94,95). Fluorescence resonance energy transfer (FRET) analysis of recombinant IP 3 Rs also showed reversible structural changes induced by physiological concentrations of IP 3 and Ca 2+ (96).…”