CALCIUM CLAMP IN ISOLATED NEURONS OF THE SNAIL HELIX-POMATIA

1. Intracellular free calcium concentration ([Ca2+]i) in isolated non-identified Helix pomatia neurones has been clamped at different physiologically significant levels by a feedback system between the fluorescent signal of fura-2 probe loaded into the cell and ionophoretic injection of Ca2+ ions through a CaCl2-loaded microelectrode. The membrane potential of the neurone has also been clamped using a conventional two-microelectrode method. 2. Special measurements have shown that the transport indices of injecting microelectrodes filled with 50 mm CaCl2 are quite variable (0.11 +/- 0.06, mean +/- S.D.). However, for each electrode the transport indices remained stable during several injection trials into a solution drop having the size of a neurone. The spread of calcium ions from the tip of the microelectrode across the cytosol of the neurone terminated within 2-4 s. The spatial difference in [Ca2+]i at this time did not exceed 10%. 3. Clamping of [Ca2+]i at a new increased level was accompanied by a transient of the Ca2+-injecting current. To increase [Ca2+]i by 0.1 muM, the amount of calcium ions injected during this stage had to be 36 +/- 20 muM Ca2+ per cell volume. Obviously, this transient represents the filling of a fast cytosolic buffer which has to be saturated to reach a new increased level of [Ca2+]i. It was followed by a steady component of Ca2+/-injecting current, which was quite low (corresponding to injection of 0.39 +/- 0.20 muM s-1 for a 0.1 muM change of [Ca2+]i). This may represent the functioning of Ca2+-eliminating systems and corresponds to a similar amount of Ca2+ extruded from the cytoplasm. 4. Changes in the injection current also developed when Ca2+ influx through the membrane was triggered by the activation of voltage-gated calcium channels. The amount of Ca2+ entering the cell during the first seconds of depolarization to - 15 mV was equal to 0-59 +/- 0.31 muM s-1 per cell volume. 5. No activation of Ca2+-dependent potassium current was observed during the changes in [Ca2+]i to levels exceeding the basal one by several times. Obviously, to activate this current, a much stronger increase in [Ca2+]i is needed in the immediate vicinity of the corresponding channels.