Analogue computation of transient changes of intracellular free Ca²⁺ concentration with the low affinity Ca²⁺ indicator furaptra during whole-cell patch-clamp recording

Abstract

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