LAUSR

Previously used techniques to measure the sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) function either are cell destructive or lack sensitivity. We designed a new approach that allows studying SERCA function in the cellular environment under well-controlled conditions. We used the genetically encoded Ca sensor CEPIA-1er to directly measure ER [Ca] ([Ca]ER) in HEK293 cells expressing SERCA2a. These cells were also expressing ryanodine receptor (RyR2) for pharmacological control of ER Ca content. The plasma membrane was permeabilized with saponin to control cytosolic [Ca] and energy supply. HEK293 cells expressing wt-SERCA2a manifested periodic [Ca]ER depletions (Ca waves) due to spontaneous activation of RyR2. Ca waves were rarely seen in cells expressing only RyR2, because endogenous SERCA was unable to increase [Ca]ER to a critical level that triggers spontaneous Ca-induced Ca release (CICR). Application of 10 mM caffeine completely depleted [Ca]ER. Once caffeine was removed, RyR2 inhibitors were applied to measure the rate of [Ca]ER recovery. Thus, ER Ca uptake can be analyzed throughout the whole physiological range of [Ca]ER. ER Ca uptake was progressively decreased with increasing cytosolic [ADP] and can be blocked by thapsigargin. At the end of each experiment, the CEPIA-1er signal was calibrated with ionomycin. Using this approach, we characterized properties of wt- and AAA-SERCA2a (partial loss-of-function SERCA2a mutant). ER Ca uptake as a function of [Ca]ER was analyzed to estimate maximum ER Ca uptake rate and maximum ER Ca load generated by wt and the SERCA mutant. We found that AAA-SERCA2a had slower Ca uptake rate than wt-SERCA2a, as a result HEK293 cells expressing AAA-SERCA2a were not effective to increase ER Ca load to the level that triggers Ca waves. Thus, this new approach can be used as a sensitive screening tool to study different drugs and mutations that affect SERCA function.