ANALOG COMPUTATION OF TRANSIENT CHANGES OF INTRACELLULAR FREE CA2-AFFINITY CA2+ INDICATOR FURAPTRA DURING WHOLE-CELL PATCH-CLAMP RECORDING(CONCENTRATION WITH THE LOW)

The measurement of cytosolic free Ca2+ ion concentration ([Ca2+]) with low affinity Ca2+ indicators has advantages for kinetic studies of cy tosolic [Ca2+] transients when compared with more commonly used high a ffinity Ca2+ indicators. Their dynamic range and linearity are better suited to measurement of high localised transient concentration change s that exist near sites of influx or release, and the additional buffe ring introduced by the indicator is minimised. The fluorescent indicat or furaptra (magfura-2) has low affinity for Ca2+ (approx. 50 mu M) an d can be used conveniently with single wavelength excitation at 420 nm with the procedure described by Konishi et al. [6]. The application o f this protocol in whole-cell patch-clamp recording permits calibrated measurements of [Ca2+] during an experiment with minimal distortion o f the time course and amplitude of [Ca2+] transients. A simple and ine xpensive analogue circuit is described for direct computation of [Ca2] from furaptra fluorescence with single wavelength excitation and emi ssion during whole-cell recording. Data are shown which compare furapt ra and fluo-3 estimates of the time course and amplitude of [Ca2+] cha nges in vascular endothelia, Purkinje neurones and hepatocytes.