Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators.
HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.
Yu
R
R
Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Hinkle
P M
PM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D013973:Thyrotropin-Releasing Hormone; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium