Kinetics of the thapsigargin-induced Ca2+ mobilisation: A quantitative analysis in the HEK-293 cell line

Abstract

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump and, when applied acutely, it initiates a Ca²⁺ mobilisation that begins with the loss of Ca²⁺ from the endoplasmic reticulum (ER) and culminates with store-operated Ca²⁺ entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca²⁺ levels using FURA-2 and the FRET-based ER Ca²⁺ sensor D1ER, respectively. Our analysis predicts an ER Ca²⁺ leak of 5-6 µM⋅s^-1 for the typical basal ER Ca²⁺ level of 335-407 µM in HEK-293 cells. The resulting cytosolic Ca²⁺ transients reached peak amplitudes of 0.6-1.0 µM in the absence of external Ca²⁺ and were amplified by SOCE that amounted to 28-30 nM⋅s^-1 in 1 mM external Ca²⁺. Additionally, cytosolic Ca²⁺ transients were shaped by a Ca²⁺ clearance of 10-13 nM⋅s^-1. Using puromycin (PURO), which enhances the ER Ca²⁺ leak, we show that TG-induced cytosolic Ca²⁺ transients are directly related to ER Ca²⁺ levels and to the ER Ca²⁺ leak. A one-compartment model incorporating ER Ca²⁺ leak and cytosolic Ca²⁺ clearance accounted satisfactorily for the basic features of TG-induced Ca²⁺ transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca²⁺ transients most likely reflects an increase in ER Ca²⁺ leak.