Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca ATPase (SERCA) pump and, when applied acutely, it initiates a Ca mobilisation that begins with the loss of Ca from the endoplasmic reticulum (ER) and culminates with store-operated Ca entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca levels using FURA-2 and the FRET-based ER Ca sensor D1ER, respectively. Our analysis predicts an ER Ca leak of 5-6 µM⋅s for the typical basal ER Ca level of 335-407 µM in HEK-293 cells. The resulting cytosolic Ca transients reached peak amplitudes of 0.6-1.0 µM in the absence of external Ca and were amplified by SOCE that amounted to 28-30 nM⋅s in 1 mM external Ca. Additionally, cytosolic Ca transients were shaped by a Ca clearance of 10-13 nM⋅s. Using puromycin (PURO), which enhances the ER Ca leak, we show that TG-induced cytosolic Ca transients are directly related to ER Ca levels and to the ER Ca leak. A one-compartment model incorporating ER Ca leak and cytosolic Ca clearance accounted satisfactorily for the basic features of TG-induced Ca transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca transients most likely reflects an increase in ER Ca leak.
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| http://dx.doi.org/10.3389/fphys.2023.1127545 | DOI Listing |
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