Surface accessibility and conformational changes in the N-terminal domain of type I inositol trisphosphate receptors: studies using cysteine substitution mutagenesis.
To identify surface-accessible residues and monitor conformational changes of the type I inositol 1,4,5-trisphosphate receptor protein in membranes, we have introduced 10 cysteine substitutions into the N-terminal ligand-binding domain. The reactivity of these mutants with progressively larger maleimide-polyethylene glycol derivatives (MPEG) was measured using a gel shift assay of tryptic fragments. The results indicate that the mutations fall into four categories as follows: sites that are highly accessible based on reactivity with the largest 20-kDa MPEG (S2C); sites that are moderately accessible based on reactivity only with 5-kDa MPEG (S6C, S7C, A189C, and S277C); sites whose accessibility is markedly enhanced by Ca(2+) (S171C, S277C, and A575C); and sites that are inaccessible irrespective of incubation conditions (S217C, A245C, and S436C). The stimulation of accessibility induced by Ca(2+) at the S277C site occurred with an EC(50) of 0.8 mum and was mimicked by Sr(2+) but not Ba(2+). Inositol 1,4,5-trisphosphate alone did not affect reactivity of any of the mutants in the presence or absence of Ca(2+). The data are interpreted using crystal structures and EM reconstructions of the receptor. Our data identify N-terminal regions of the protein that become exposed upon Ca(2+) binding and suggest possible orientations of the suppressor and ligand-binding domains that have implications for the mechanism of gating of the channel.
Anyatonwu
Georgia
G
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Joseph
Suresh K
SK
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D008670:Metals / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011092:Polyethylene Glycols / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats
D053496:Inositol 1,4,5-Trisphosphate Receptors; D008670:Metals; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D003545:Cysteine