Direct association of ligand-binding and pore domains in homo- and heterotetrameric inositol 1,4,5-trisphosphate receptors.
Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of intracellular Ca(2+) channels that exist as homo- or heterotetramers. In order to determine whether the N-terminal ligand-binding domain is in close physical proximity to the C-terminal pore domain, we prepared microsomal membranes from COS-7 cells expressing recombinant type I and type III IP(3)R isoforms. Trypsin digestion followed by cross-linking and co-immunoprecipitation of peptide fragments suggested an inter-subunit N- and C-terminal interaction in both homo- and heterotetramers. This observation was further supported by the ability of in vitro translated C-terminal peptides to interact specifically with an N-terminal fusion protein. Using a (45)Ca(2+) flux assay, we provide functional evidence that the ligand-binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP(3)Rs and propose that the gating mechanism of IP(3)R Ca(2+) channels involves the association of the N-terminus of one subunit with the C-terminus of an adjacent subunit in both homo- and heterotetrameric complexes.
Boehning
D
D
Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, PA 19107, USA.
Joseph
S K
SK
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D003432:Cross-Linking Reagents / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating / Q000187:drug effects; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D015220:Calcium Channels; D003432:Cross-Linking Reagents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D000644:Quaternary Ammonium Compounds; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D013388:Succinimides; C034730:zwittergent 3-12; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D015544:Inositol 1,4,5-Trisphosphate; D014357:Trypsin; D002118:Calcium