Regulation of inositol trisphosphate receptors

肌醇三磷酸受体的调节

• 批准号: 10326833
• 负责人: SURESH K JOSEPH
• 金额: $ 32.18万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326833
• 关键词: Ablation; Address; Adenine Nucleotides; Agonist; Amoeba genus; Animal Model; Binding; Binding Sites; Bioenergetics; Biological; C-terminal; CRISPR/Cas technology; Calcium; Calcium Signaling; Carbon; Cell Cycle; Cell Death; Cell Line; Cell division; Cell membrane; Cell physiology; Cells; Cervix carcinoma; Charge; Chemicals; Chickens; Citric Acid Cycle; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cryoelectron Microscopy; D Cells; Dermal; Diabetes Mellitus; Disease; Drug or chemical Tissue Distribution; Endoplasmic Reticulum; Energy Supply; Enzymes; Exocytosis; FRAP1 gene; Family; Feedback; Fertilization; Fibroblasts; Frequencies; Gene Expression; Genetic; Genetic Transcription; Glucose; Glutamine; Glycolysis; Goals; Growth; Growth Factor; Heart Diseases; Hela Cells; Hepatocyte; Hereditary Disease; Homo; Hormones; Human Cell Line; ITPR1 gene; Inositol; Interruption; Knock-out; Knockout Mice; Ligand Binding Domain; Ligands; Location; Malate-Aspartate Shuttle Pathway; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mass Spectrum Analysis; Measures; Mediating; Memory; Metabolic; Metabolism; Mitochondria; Molecular; Molecular Conformation; Monitor; Muscle Contraction; Mutagenesis; Mutate; Mutation; N-terminal; NADH; Neurodegenerative Disorders; Neurotransmitters; Normal Cell; Oxidoreductase; Oxygen Consumption; Patients; Periodicity; Phase; Phosphorylation; Phosphotransferases; Process; Production; Property; Protein Isoforms; Protein Kinase; Proteins; Regulation; Respiratory Chain; Role; Signal Transduction; Site; Staging; Structure; Technology; Testing; Therapeutic; Tracer; Transfection; Transmembrane Domain; Work; base; biological research; cancer cell; cell motility; cell transformation; crosslink; design; experimental study; extracellular; receptor; response; transcription factor

Calcium is a universal messenger controlling a multitude of cellular responses including muscle contraction, exocytosis, memory, fertilization, metabolism, proliferation and cell death. Numerous hormones, neurotransmitters and growth factors stimulate the formation of inositol 1,4,5-trisphosphate ( IP3 ) which acts on a family of receptors ( IP3Rs ) located in the endoplasmic reticulum that function as ligand-gated Ca2+ channels. The depletion of intracellular Ca2+ stores also activates Ca2+ influx mechanisms in the plasma membrane. Thus, the interaction of IP3 with its receptor activates all phases of a Ca2+ signal. IP3Rs are regulated by Ca2+ and phosphorylation but the molecular basis of this regulation is poorly understood. Ca2+ released from IP3Rs is locally transmitted to the mitochondria and can stimulate metabolism, and in higher amounts, can also initiate cell death. Cancer cells have been proposed to be highly dependent on IP3R- mediated mitochondrial Ca2+ transfer for their survival. The advent of CRISPR/Cas-9 technology has allowed the genetic ablation of all three IP3R isoforms from HEK293 and HeLa cervix carcinoma cells. The proposal is centered on the use of these cell lines to a) study the adaptive mechanisms allowing the cells to survive in the complete absence of Ca2+ signaling and b) to take advantage of a null background for structure-function studies exploring the molecular mechanism by which IP3 opens the channel and the mechanism of feed-back regulation by Ca2+. The two specific aims are: 1] Characterize adaptive mechanisms in IP3R-3KO cells: The impact of a lack of Ca2+ regulation of metabolism will be investigated by measuring several bioenergetic parameters and 13C-tracer metabolism. Proliferation, cell-cycle status, cell death and transcriptional rewiring will be explored. 2A] How does IP3 open the channel? Chemical crosslinking/mass spectroscopy will be used to validate cryo-EM structures and monitor conformational changes mediated by IP3 in native IP3Rs. Mutagenesis of key residues will be used to test proposed allosteric mechanisms of gating. 2B] Identification of the Ca2+ regulatory sites in IP3Rs: Based on the most recent cryo-EM structures of IP3R1 and IP3R3 we have identified 3 clusters of negatively charged residues that are candidates for Ca2+ regulatory sites. Their functional role as stimulatory or inhibitory sites will be assessed by mutagenesis. The role of these sites will also be tested by mutagenesis of the Ca2+-insensitive IP3R from Capsaspora owczarzaki . The long-term goal of the proposal is to better understand the role of Ca2+signaling in normal cells, cancer cells and in inherited disorders inactivating IP3R function. The study should also provide fundamental mechanistic information on how these important channels work and are regulated.

钙是一种控制多种细胞反应的通用信使，包括肌肉收缩，