Critical regions for activation gating of the inositol 1,4,5-trisphosphate receptor.
To understand the molecular mechanism of ligand-induced gating of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)/Ca(2+) release channel, we analyzed the channel properties of deletion mutants retaining both the IP(3)-binding and channel-forming domains of IP(3)R1. Using intrinsically IP(3)R-deficient cells as the host cells for receptor expression, we determined that six of the mutants, those lacking residues 1-223, 651-1130, 1267-2110, 1845-2042, 1845-2216, and 2610-2748, did not exhibit any measurable Ca(2+) release activity, whereas the mutants lacking residues 1131-1379 and 2736-2749 retained the activity. Limited trypsin digestion showed that not only the IP(3)-gated Ca(2+)-permeable mutants lacking residues 1131-1379 and 2736-2749, but also two nonfunctional mutants lacking residues 1-223 and 651-1130, retained the normal folding structure of at least the C-terminal channel-forming domain. These results indicate that two regions of IP(3)R1, viz. residues 1-223 and 651-1130, are critical for IP(3)-induced gating. We also identified a highly conserved cysteine residue at position 2613, which is located within the C-terminal tail, as being essential for channel opening. Based on these results, we propose a novel five-domain structure model in which both N-terminal and internal coupling domains transduce ligand-binding signals to the C-terminal tail, which acts as a gatekeeper that triggers opening of the activation gate of IP(3)R1 following IP(3) binding.
Uchida
Keiko
K
Department of Basic Medical Science, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Miyauchi
Hiroshi
H
Furuichi
Teiichi
T
Michikawa
Takayuki
T
Mikoshiba
Katsuhiko
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D017124:Conserved Sequence; D003545:Cysteine; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating / Q000235:genetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000502:physiology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D003545:Cysteine