Intracellular synthesis of secreted and cell surface proteins takes place in a membraneous network called the endoplasmic reticulum (ER). The physiological condition of deregulated homeostasis in the lumen of the ER is commonly referred to as ER stress. It is now increasingly recognized that ER stress-evoked calcium transmission from the ER to another membrane-bound cell organelle, the mitochondrium, can impart and amplify a cell death signal. This process involves a complicated network of signaling cascades. In most non-muscle tissues, calcium signals emanating from the ER depend on calcium pumping into the ER by sarco(endo)plasmic reticulum calcium ATPase isoform 2b (SERCA2b) and calcium release through inositol 1,4,5-trosphosphate receptor (IP3R) channel proteins. Here, we hypothesize a regulatory mechanism that integrates oxidative stress in the ER into the propagation of death signaling pathways via redox-dependent modulation of SERCA2b and/or IP3R activity. Both of these ER-resident multispanning membrane proteins comprise a conserved pair of luminal cysteines. To investigate if ER environment-dependent thiol-disulfide conversion of these cysteines may represent a regulatory redox switch, we will establish a quantitative procedure to determine their in vivo redox state. Because both SERCA2b and IP3Rs are very large proteins comprising many cysteines, different strategies to trim the polypeptides and isolate the fragments harboring the luminal peptides will be applied. In a next step, we will search for conditions that impact the in situ thiol-disulfide distribution of the presumable switch cysteines. To achieve this, we will study their redox modulation induced both by over-expression or knockdown of selected ER proteins and by treatment of the cells with different concentrations of reductants and oxidants as well as with general inducers of ER stress. Having established conditions of most specific SERCA2b and IP3R thiol-disulfide manipulation, we will test the functionality of the redox switches by analyzing the activity of SERCA2b and IP3Rs in treated versus control cells. In parallel experiments, the mechanistic aspect of redox switch catalysis will be investigated by hunting mixed-disulfide interaction partners of the SERCA2b and IP3Rs luminal cysteines. Overall, the anticipated results are expected to enable a more profound comprehension of the processes that determine cell viability during physiological situations of stress. Such comprehension is of crucial importance and will aid the targeted treatment of devastating diseases such as neurodegenerative disorders and cancer.