Mechanism of calcium release during unfolded protein respose

HOLSTEIN D.; LECHLEITER J.; FELIZIANI C.; PATON A. W.; BOLLO M.; QUASSOLLO G.; PATON J.C.

Córdoba

Congreso; XXXIII Reunión Anual de la Sociedad Argentina de Neurociencias; 2018

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 reflectsa balance between active uptake by Ca2+-ATPases and passive efflux pathways of which the translocon can play a prominent role. The translocon is an aqueous pore, primarily formed by the Sec61 core spanning the ERlipid 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 afteraccumulation of unfolded protein in the lumen, the Ca2+ ER eflux through the translocon is increased. To test this mechanism, 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 thatwas 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 thetranslocon 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 ribosomalprotein S6. Important to note that the likehood of obtained Tm-induce local Ca2+ events, increase by using either the slow chelator EGTA-AM or Xestospongin C and Ryanodine (InsP3and Ry Receptors inhibitors,respectively). Moreover, we specifically plug the pore using 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-tRNAblocking the pore. The Prl-64 expression clearly exhibited Tm-induce Ca2+local increase. Finally, InsP3 Receptor null HEK-293 cell line (HEK3KO) exhibited Tm-evoked Ca2+release near ER microdomain, which is inhibited byBiP over-expression. 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