ENDOPLASMIC RETICULUM STRESS INDUCES CA2+ RELEASE

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

Congreso; Calcium Signalling Gordon Research Conference; 2017

The Endoplasmic Reticulum (ER) is amulti-functional organelle that plays a critical role in a variety ofprocesses, 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 whichthe translocon can play a prominent role (J Biol Chem, 29:26479; J Cell Sci. 117: 4135 Pflugers Arch - EurJ Physiol 453:797). The translocon is an aqueous pore, primarily formedby the Sec61a core spanning the ER lipid bilayer, that is blocked by the ribosome on thecytosolic side and by the ER chaperone, BiP, on the luminal side. Wehypothesize that during the acute phase of the UPR (Unfolded ProteinResponse), immediately after accumulation of unfolded proteinin the lumen, the Ca2+ER eflux through the translocon isincreased. 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 transloconblockers (emetine or anisomycin). Inaddition, the Tm-induced Ca2+ signal was amplified by pre-treatment either with AB5cytotoxine, which specifically hydrolyses BiP, or with the translocon opener puromycin. The effectof these pharmacological tools was corroborated by co-immunoprecipitations thatshowed changes in the interactions either between Sec61α and BiP or Sec61α andthe ribosomal protein S6. Important to note thatthe likehood of obtained Tm-induce local Ca2+ events, increase byusing either the slow chelator EGTA-AM orXestospongin C and Ryanodine (InsP3 and Ry Receptorsinhibitors, respectively). Moreover, we specifically plug the pore using atruncated  prolactin with 64 amino acidresidues lacking the stop codon (Prl-64), thus the nascent chain remains boundto the ribosome as a peptidyl-tRNA blocking the pore. The Prl-64 expressionclearly inhibed Tm-indude Ca2+ local increase. Taken together, these data,strongly suggest that the chaperoneBiP and the ribosome are dissociated from the translocon increasing Ca2+permeability.  Funded in part by NIH R21NS085732.