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 Ca2+ indicators designed to measure the free Ca2+ concentration in the cytoplasm, [Ca2+](cyt), and the endoplasmic reticulum (ER), [Ca2+](er). Basal [Ca2+](cyt) was about 50 nM; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca2+](cyt) to 1 mu M or higher. Basal [Ca2+](er) averaged 500 mu M and fell to 50-100 mu M over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca2+](er) to 100 mu M, independent of extracellular Ca2+, whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca2+](er) to 5-10 mu M, indicating that there is substantial store refilling during signaling. TRH increased [Ca2+](cyt) and decreased [Ca2+](er) if applied after other agonists, whereas other agonists did not alter [Ca2+](cyt) or [Ca2+](er) if added after TRH. When Ca2+ was added back to cells that had been incubated with TRH in Ca2+-free medium, [Ca2+](cyt) and [Ca2+](er) increased rapidly. The increase in [Ca2+](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. acid. in conclusion, these new Ca2+ indicators showed that basal [Ca2+](er) is similar to 500 mu M, that [Ca2+](er) has to be > 100 mu M to support an increase in [Ca2+](cyt) by agonists, and that during signaling, intracellular Ca2+ stores are continuously refilled with cytoplasmic Ca2+ by the sarcoendoplasmic reticulum Ca2+-ATPase pump.