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

Frontiers in Physiology

Physiology

René Tinschert¹  Igor Gamayun¹  Tillman Pick²  Adolfo Cavalié³ 

[1] Experimental and Clinical Pharmacology and Toxicology, Pre-clinical Center for Molecular Signalling (PZMS), Saarland University, Homburg, Germany;[e-mail];null;

关键词: Ca homeostasis;    Ca leak;    store-operated Ca entry;    Ca imaging;    thapsigargin;   

DOI  :  10.3389/fphys.2023.1127545

 received in 2022-12-19, accepted in 2023-03-07,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Pick, Gamayun, Tinschert and Cavalié.