Kinetics of the thapsigargin-induced Ca(2+) mobilisation: A quantitative analysis in the HEK-293 cell line

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump and, when applied acutely, it initiates a Ca(2+) mobilisation that begins with the loss of Ca(2+) from the endoplasmic reticulum (ER) and culminates with store-operated Ca(2+) entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca(2+) levels using FURA-2 and the FRET-based ER Ca(2+) sensor D1ER, respectively. Our analysis predicts an ER Ca(2+) leak of 5–6 µM⋅s(−1) for the typical basal ER Ca(2+) level of 335–407 µM in HEK-293 cells. The resulting cytosolic Ca(2+) transients reached peak amplitudes of 0.6–1.0 µM in the absence of external Ca(2+) and were amplified by SOCE that amounted to 28–30 nM⋅s(−1) in 1 mM external Ca(2+). Additionally, cytosolic Ca(2+) transients were shaped by a Ca(2+) clearance of 10–13 nM⋅s(−1). Using puromycin (PURO), which enhances the ER Ca(2+) leak, we show that TG-induced cytosolic Ca(2+) transients are directly related to ER Ca(2+) levels and to the ER Ca(2+) leak. A one-compartment model incorporating ER Ca(2+) leak and cytosolic Ca(2+) clearance accounted satisfactorily for the basic features of TG-induced Ca(2+) transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca(2+) transients most likely reflects an increase in ER Ca(2+) leak.