Focal calcium release during acute unfolded protein response

FELIZIANI C; DEBORAH HOLSTEIN; QUASOLLO G., ; PATON A.W., ; PATON J.C; LECHLETER J; BOLLO M

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

The Endoplasmic Reticulum (ER) is a multi-functional organelle that plays a critical role in a variety of processes, where the ER Ca2+ acts as a key messenger. Under resting conditions, the luminal Ca2+ concentration reflects a balance between active uptake by Ca2+-ATPases and passive efflux pathways of which the translocon can play a prominent role (J Biol Chem, 29:26479; J Cell Sci. 117: 4135 Pflugers Arch - Eur J Physiol 453:797). The translocon is an aqueous pore, primarily formed by the Sec61α core spanning the ER lipid bilayer, that is blocked by the ribosome on the cytosolic side and by the ER chaperone, BiP, on the luminal side. We hypothesize that during the acute phase of the UPR (Unfolded Protein Response), immediately after accumulation of unfolded protein in the lumen, the Ca2+ ER eflux through the translocon is increased. To test this mechanism of action, we performed cytosolic Ca2+ measurements in primary cultures of human astrocytes, expressing the Ca2+ indicator GCaMP6 tethered to the ER membrane, after induction of the UPR by Tunicamycin (Tm, glycosylation inhibitor). We observed focal release of Ca2+ (microdomains) in stressed astrocytes that was significantly inhibited by translocon blockers (emetine or anisomycin). In addition, the Tm-induced Ca2+ signal was amplified by pre-treatment either with AB5 cytotoxine, which specifically hydrolyses BiP, or with the translocon opener puromycin. The effect of these pharmacological tools was corroborated by co-immunoprecipitations that showed changes in the interactions either between Sec61α and BiP or Sec61α and the ribosomal protein S6. Moreover, we specifically plug the pore by expressing a truncated prolactin with 64 amino acid residues lacking the stop codon (Prl-64), thus the nascent chain remains bound to the ribosome as a peptidyl-tRNA blocking the pore. The Prl-64 expression clearly inhibited Tm-indude Ca2+ local increase. Taken together, these data, strongly suggest that the chaperone BiP and the ribosome are dissociated from the translocon increasing Ca2+ permeability. Funded in part by NIH R21 NS085732.