The use of fura-2 for estimating Ca buffers and Ca fluxes.
The compound fura-2 (Grynkiewicz et al., J. Biol. Chem. 260, 3440-3450, 1985) is generally known as an indicator dye for measuring the concentration of free calcium ([Ca2+]) inside living cells. It should be appreciated, however, that this is not what it actually is. More accurately, it is a divalent metal ion chelator which changes its fluorescence properties upon complexation. Thus, [Ca2+] has to be inferred indirectly by means of the law of mass action. As a chelator, fura-2 may influence the quantity of interest, the Ca signal. On the other hand, the chelator action may be used for a number of other purposes, some of them more directly related to its molecular properties: as a chelator, competing with endogenous Ca buffers, it can be used to estimate endogenous buffers and their properties. When present at sufficiently high concentration, such that it outcompetes endogenous buffers, fura-2 reports total Ca changes and is a probe for Ca fluxes across the membrane. Here, theory and methodological considerations of such applications of fura-2 will be summarized and results on Ca buffer and Ca flux measurements derived from various methods will be compared.
Neher
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Max-Planck-Institut für biophysikalische Chemie, Department of Membranebiophysics, Göttingen, Germany.
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D002468:Cell Physiological Phenomena; D002614:Chelating Agents; D005456:Fluorescent Dyes; D016257:Fura-2; D008433:Mathematics; D013050:Spectrometry, Fluorescence / Q000379:methods
D002021:Buffers; D002614:Chelating Agents; D005456:Fluorescent Dyes; D002118:Calcium; D016257:Fura-2