Mechanism of Ca2+ Release During Acute Phase Of Endoplasmic Reticulum Stress

FELIZIANI C.; HOLSTEIN D.; LECHLEITER J.; BOLLO M.

Mar Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

The Endoplasmic Reticulum(ER) is a multi-functional organelle that plays a critical role in a variety ofprocesses, such as Ca2+ storage and release, synthesis and foldingof proteins and post-translational protein modification. These processes aredeeply interconnected, where the ER Ca2+ acts as a link.                 Underresting conditions, the ER intraluminal free calcium concentration reflects abalance between active uptake by Ca2+-ATPases and passive efflux via leak channels.  Have been proposed that a basal Ca2+ leak occur via translocon.This is an aqueous pore that completely spans the ER lipid bilayer throughwhich newly synthesized secretory proteins are translocated. The heteromeric Sec61 complex forms the coreof this pore, which is blocked by the ribosome on the cytosolic side and bythe ER chaperone, BiP, on the luminal side. Our hypothesisestablishes that during the acute phase of UPR (Unfolded Protein Response),immediately after accumulation of unfolded protein in the ER, the Ca2+ER permeability increase through the translocon. The aim of this project is to delineate themechanism of Ca2+ release during acute phase of EndoplasmicReticulum stress.Using primary mouse culture astrocytes we found that Tunicamycin (Tm, glycosylation inhibitor, that triggers an overallincrease cytosolic Ca2 +) induced Ca2+ release inmicrodomains near the ER. To maximize thesensitivity of these measurements we used a genetically encoded Ca2+indicator that is tethered to the ER membrane. We also obtained pharmacological evidence that the translocon may be thesource of this Ca2+ leak. We found that astrocytes pre-treated withemetine (a translocon inhibitor) significantly inhibit the Tm-induced increasein cytosolic Ca2+ , however astrocytespre-treated with AB5 cytotoxine (which specifically hydrolyzes BiP from the translocon luminal side) increase Ca2+ release. These results was confirmed when the astrocites werepre-treated with anisomycin (an inhibitor of the peptdil transferasas) and thetranslocon opener puromycin  (atranslation inhibitor that specifically releases polypeptide chains).