title: Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers.
abstract: Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085, USA.
authors.forename: M
authors.initials: M
authors.lastname: Zhao
authors.forename: S
authors.initials: S
authors.lastname: Hollingworth
authors.forename: S M
authors.initials: SM
authors.lastname: Baylor
mesh_terms: D000200:Action Potentials; D000818:Animals; D001572:Benzofurans; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007093:Imidazoles; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007211:Indoles; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D009930:Organic Chemicals; D011896:Rana temporaria; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001572:Benzofurans; D005456:Fluorescent Dyes; D007093:Imidazoles; D007202:Indicators and Reagents; D007211:Indoles; D009930:Organic Chemicals; C094537:mag-Fura 5; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; C088692:2-(4-(biscarboxymethyl)amino-3-(carboxymethoxy)phenyl)-1H-indole-6-carboxylic acid; C071336:calcium green; C071335:calcium orange; C082877:fura red; D002118:Calcium; D016257:Fura-2