In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling 1 . 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 [2][3][4][5][6][7][8] . 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 9,10 . 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 [11][12][13] . This system combines siRNA mediated gene silencing and live cell Ca 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.
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Preparation of stock solutions1. Prepare calcium-free buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 0.5 mM EGTA, 10 mM glucose in 10 mM HEPES-KOH, (pH 7.35)). 2. Solubilize FURA-2 AM in DMSO to obtain an 1 mM stock solution. Mix using a vortex until the solution is homogenously light yellow. Protect cup from light. Dilute the FURA-2 AM stock solution into 1 ml Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to a final concentration of 4 μM for loading of HeLa cells. 3. Prepare a 12.5 mM puromycin stock solution in distilled water (pH 7.0). Store aliquots at -20°C. Dilute the 12.5 mM puromycin stock solution in the calcium-free buffer to a final concentration of 500 μM puromycin. 4. Dissolve thapsigargin in DMSO to obtain a 1 mM Stock solution. 5. Solubilize 1 mg ophiobolin A in 20 μl chloroform and, subsequently, mix with 25 μl DMSO. The microcentrifuge tube remains open until chloroform is evaporated. Thus the final ophiobolin A concentration is 100 mM. Store aliquots at -20 °C. Prior to use, dilute stock solutions into calcium-free buffer to a final concentration of 100 μM. Thus, final DMSO concentration for live cell imaging does not exceed 0.1%. 6. Dissolve trifluoperazine in DMSO to a concentration of 10 mM and store aliquots at -20 °C. Dilute stock solutions into calcium-free buffer to a final concentration of 10 μM prior to use. Thus final DMSO con...