Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.

Abstract:

: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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Westerblad H,Allen DG

doi

10.1016/S0006-3495(96)79294-9

subject

Has Abstract

pub_date

1996-08-01 00:00:00

pages

908-17

issue

2

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(96)79294-9

journal_volume

71

pub_type

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