Journal: PLoS Genetics
Article Title: Absence of the ER Cation Channel TMEM38B /TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta
Figure Lengend Snippet: Secreted proteins such as collagen are synthesized within the ER, which contains the major intracellular Ca 2+ store. In response to extracellular stimuli, Ca 2+ is released from the ER lumen into the cytoplasm via IP 3 and ryanodine receptors. Cytoplasmic Ca 2+ that does not bind signaling proteins involved in gene expression is transported back into the ER via SERCA pumps. The continuous fluctuations in free [Ca 2+ ] to and from the ER are indirectly regulated by TRIC channel activity, which mediates transmembrane K + flux to maintain electroneutrality across the ER membrane. In fibroblasts and osteoblasts, ER-resident Ca 2+ -binding chaperones, including BiP, CyPB, PDI and calreticulin (CRT), directly interact with collagen alpha chains (red polypeptides) to facilitate or catalyze specific modifications required for assembly and folding. In the absence of TRIC-mediated K + flux, Ca2 + -dependent “cycling” of these chaperones between inactive, sequestered forms and active, substrate-interacting forms, is dysregulated. Thus, absence of TRIC-B alters ER luminal [Ca 2+ ], affecting synthesis and secretion of collagen, as well as interactions of collagen-specific chaperones with each other and with their substrate, resulting in abnormal collagen assembly and post-translational modification. BiP, GRP78; CyPB, cyclophilin B; CRT, calreticulin; IP3R, inositol triphosphate receptor; LH1, lysyl hydroxylase 1; PDI, protein disulfide isomerase; SERCA2b, sarcoplasmic-endoplasmic reticulum Ca 2+ -ATPase isoform 2b; TRIC-B, trimeric intracellular cation channel subtype B.
Article Snippet: Gene transcript levels were quantitated by real-time RT-PCR following reverse-transcription using a High Capacity cDNA Archive Kit and Taqman Assays on Demand (Life Technologies, TMEM38A , Hs00225325_m1; TMEM38B , Hs00216531_m1; COL1A1 , Hs00164004_m1; FKBP10 , Hs01000263_m1; PLOD1 , Hs00609368_m1; PLOD2 , Hs01118190_m1; PLOD3 , Hs01126617_m1; P4HB/PDI , Hs00168586_m1; HSPA5 , Hs99999174_m1; PPIB , Hs00168719_m1; ATP2A/SERCA2 , Hs00544877_m1; ITPR1 , Hs00181881_m1; RYR1 , Hs00166991_m1; GAPDH , Hs99999905_m1; ACTB , Hs99999903_m1; B2M , Hs99999907_m1).
Techniques: Synthesized, Expressing, Activity Assay, Binding Assay, Modification