Calcium-dependent conformational changes in inositol trisphosphate receptors.
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 approximately 0.5 microm 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.
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Georgia
G
Anyatonwu
M Tariq
MT
Khan
Zachary T
ZT
Schug
Paula C A
PC
da Fonseca
Edward P
EP
Morris
Suresh K
SK
Joseph
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry*; D008862:Microsomes, Liver / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000939:Epitopes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D003545:Cysteine; D002118:Calcium