CALCIUM CLAMP IN SINGLE NERVE-CELLS

Free calcium concentration in isolated single neurons was clamped usin g a new technical approach based on a feed-back connection between the Fura-2 fluorescence signal measuring the intracellular Ca2+ concentra tion ([Ca2+]i) and iontophoretic current injecting Ca2+ into the cell. Beginning of [Ca2+]i clamping at a level above the basal one triggere d fast (few seconds) current transients equal to injection of 36 +/- 2 0 muM Ca2+ (for a 0.1 muM change of [Ca2+]i), representing the filling of a fast cytosolic buffer. Continuation of clamping required very sm all clamping currents (corresponding to injection of 0.39 +/- 0.20 muM .s-1 Ca2+). This value increased proportionally to the magnitude of th e change of [Ca2+]i above basal level, indicating the activation of ca lcium-dependent mechanisms for Ca2+ removal from the cytosol. The desc ribed approach allowed measurement, under physiological conditions, of the capacitative and kinetic properties of different Ca-regulating sy stems functioning in a single nerve cell as well as other types of cel ls.