title: Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor.
abstract: Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9040, USA.
authors.forename: Huiping
authors.initials: H
authors.lastname: Tu
authors.forename: Elena
authors.initials: E
authors.lastname: Nosyreva
authors.forename: Tomoya
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Zhengnan
authors.initials: Z
authors.lastname: Wang
authors.forename: Akiko
authors.initials: A
authors.lastname: Mizushima
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Iino
authors.forename: Ilya
authors.initials: I
authors.lastname: Bezprozvanny
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera / Q000737:chemistry / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D002118:Calcium