Calcium homeostasis in the presence of fura-2 in neurons dissociated from rat nucleus basalis: theoretical and experimental analysis of chelating action of fura-2.

Intracellular calcium ions (Ca2+) play important roles in cell functions. Measurements of intracellular calcium ion concentration ([Ca2+]i) are often made with the fura-2 fluorescence recording technique in various preparations including neurons. Fura-2 has, however, a Ca(2+)-chelating action which complicates the interpretation of experimental results. In this report the chelating action of intracellular fura-2 was studied by means of computer simulations. The chelating action of an endogenous Ca(2+)-binding protein, calmodulin, was also estimated. Furthermore, whole-cell patch-clamp recordings of calcium currents (ICa) and fura-2 microfluorimetric recordings of [Ca2+]i were simultaneously made from neurons which were acutely dissociated from the rat nucleus basalis. Since Ca2+ influx can be initiated and terminated by using the voltage-clamp technique, the relationship between Ca2+ influx and rapid [Ca2+]i increase was examined. The present theoretical evaluations and experimental results disclosed the relationship between fura-2 and endogenous Ca(2+)-binding proteins; fura-2 at low concentration (10 microM) did not substantially affect the endogenous Ca2+ buffering mechanisms, but at high concentration (200 microM) effectively buffered cytosolic Ca2+ instead of endogenous Ca2+ buffers. Calcium homeostasis in neurons is furthermore discussed.