Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Abstract

Intact frog skeletal muscle fibers were injected with the Ca 2+ indicator fura-2 conjugated to high molecular weight dextran (fura dextran, MW ,,d0,000; dissociation constant for Ca 2+, 0.52 p,M), and the fluorescence was measured from cytoplasm (17~ The fluorescence excitation spectrum of fura dextran measured in resting fibers was slighdy red-shifted compared with the spectrum of the Ca2+-free indicator in buffer solutions. A simple comparison of the spectra in the cytoplasm and the in vitro solutions indicates an apparently "negative" cytoplasmic [Ca2+], which probably reflects an alteration of the indicator properties in the cytoplasm. To calibrate the indicator's fluorescence signal in terms of cytoplasmic [Ca2+], we applied [3-escin to permeabilize the cell membrane of the fibers injected with fura dextran. After treatment with 5 ~M fl-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca 2+, ATP), whereas the 10-kD fura dextran only slowly leaked out of the fiber. It was thus possible to estimate calibration parameters in the indicator fluorescence in the fibers by changing the bathing solution [Ca 2+] to various levels; the average values for the fraction of Ca2+-bound indicator in the resting fibers and the dissociation constant for Ca 2+ (KD) were, respectively, 0.052 and 1.0 I~M. For the comparison, the KD value was also estimated by a kinetic analysis of the indicator fluorescence change after an action potential stimulation in intact muscle fibers, and the average value was 2.5 wM. From these values estimated in the fibers, resting cytoplasmic [Ca 2+ ] in frog skeletal muscle fibers was calculated to be 0.06-0.14 ~M. The range lies between the high estimates from other tetracarboxylate indicators (0.1-0.3 ~M; Kurebayashi, N., A.

Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells

“…Fluorescence values acquired from the images taken with excitation at both 340·nm and 380·nm for all 11 Ca 2+ buffer solutions were entered into a computer program available on the Molecular Probes website (www.probes.com/resources/calc/kd.html) to determine the value of KD. This value for KD is consistent with other in vitro estimates for fura dextran (Konishi and Watanabe, 1995;Tombal et al, 1999). The values for Rmin, Rmax, Fo and Fs were determined in situ using ciliary cells loaded with fura dextran.…”

Ionized Intracellular Calcium Concentration Predicts Excitotoxic Neuronal Death: Observations with Low-Affinity Fluorescent Calcium Indicators

“…C ytosolic calcium determination was performed with fluorescent calcium indicators: f ura-2/AM ester, f ura-2/ K ϩ salt, f ura-2 coupled to dextran [f ura-2/dextran; molecular weight (MW) 3000], and benzothiazole coumarin (BTC) (Iatridou et al, 1994;Konishi and Watanabe, 1995). All indicators were purchased from Molecular Probes (Eugene, OR).…”

Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments

“…Thus it might potentially be used for calibration of Na + , K + , Ca 2+ and Cl − sensitive probes in different cell types. This compound has been successfully used to calibrate the signal of Fura-2 for determination of intracellular calcium in frog skeletal muscle (Konishi and Watanabe, 1995b) and it was applied in our study for Cl-Sensor calibration.…”