Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.
Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.
Westerblad
H
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Department of Physiology, University of Sydney, Australia. hakan.westerblad@fyfa.ki.se
Allen
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DG
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002614:Chelating Agents; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D007211:Indoles; D007700:Kinetics; D008839:Microelectrodes; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D014982:Xenopus laevis
D002614:Chelating Agents; D005456:Fluorescent Dyes; D007211:Indoles; D004533:Egtazic Acid; C048960:indo-1; D002118:Calcium