In situ calibration and [H+] sensitivity of the fluorescent Na+ indicator SBFI

Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to mea sure intracellular free Na+ concentrations ([Na+](i)), the in situ calibrat ion techniques described to date do not favor the straightforward determina tion of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440-3450, 1985) to convert th e ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a "full" in situ calibration are fit by a thre e-parameter hyperbolic equation; the apparent dissociation constant (K-d) o f SBFI for Na+ can then be resolved by means of a three-parameter hyperboli c decay equation. We also developed and tested a "one-point" technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at th e end of an experiment during exposure to gramicidin D and 10 mM Na+ is use d as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+](i) using a modification of the stan dard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Althoug h the K-d of SBFI for Na+ was relatively insensitive to changes in pH in th e range 6.8-7.8, acidification resulted in an apparent decrease, and alkali nization in an apparent increase, in [Na+](i) values. The magnitudes of the apparent changes in [Na+](i) varied with absolute [Na+](i), and a method w as developed for correcting [Na+](i) values measured with SBFI for changes in intracellular pH.