Luminal Ca2+ dynamics were measured using the Ca2+-sensitive FRET-based cameleon protein D1-ER (76). Cells were cultured on 24-mm coverslips and transfected with D1-ER. Cells were imaged on a Zeiss Axiovert 200M microscope, equipped with a 40× oil objective (NA, 1.3). Emission ratio imaging of D1-ER was accomplished by using a 436DF20 excitation filter, a 450-nm dichroic mirror, and two emission filters [475/40 for enhanced cyan fluorescent protein (ECFP) and 535/25 for citrine]. Exposure times were 100 ms, and images were taken every 1 to 2 s. The FRET signal (yellow fluorescent protein/CFP) was normalized to CFP emission intensity, and changes in ER Ca2+ were expressed as the ratio of the emissions at 535 and 470 nm. To induce Ca2+ release from ER, the cells were challenged with an agonist that, through interaction with G protein–coupled receptors, evokes a rapid discharge from IP3Rs.

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