title: Identification of functionally critical residues in the channel domain of inositol trisphosphate receptors.
abstract: We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP(3)R) channel function. The residues selected were highly conserved in all three IP(3)R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pulldown assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP(3)R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating.
authors.affiliation: Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Cunnigaiper
authors.initials: C
authors.lastname: Bhanumathy
authors.forename: Paula C A
authors.initials: PC
authors.lastname: da Fonseca
authors.forename: Edward P
authors.initials: EP
authors.lastname: Morris
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Joseph
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D008958:Models, Molecular; D020125:Mutation, Missense; D055503:Protein Multimerization / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors