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 Ca2+ (S171C, S277C, and A575C); and sites that are inaccessible irrespective of incubation conditions (S217C, A245C, and S436C). The stimulation of accessibility induced by Ca2+ at the S277C site occurred with an EC50 of 0.8 mu M and was mimicked by Sr2+ but not Ba2+. Inositol 1,4,5-trisphosphate alone did not affect reactivity of any of the mutants in the presence or absence of Ca2+. 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 Ca2+ binding and suggest possible orientations of the suppressor and ligand-binding domains that have implications for the mechanism of gating of the channel.