Abstract
In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca(2+) leak channels. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca(2+) from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca(2+) leak channel and to characterize its putative regulatory mechanisms. This system combines siRNA mediated gene silencing and live cell Ca(2+) imaging. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca(2+)-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca(2+) concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca(2+) also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca(2+) leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca(2+) efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca(2+) efflux, and iii) study the effects of disease linked mutations in the relevant components.