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Calcium clamp in isolated neurones of the snail Helix pomatia.
Author(s) -
Belan P,
Kostyuk P,
Snitsarev V,
Tepikin A
Publication year - 1993
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1993.sp019542
Subject(s) - helix pomatia , microelectrode , calcium , chemistry , biophysics , voltage clamp , membrane potential , analytical chemistry (journal) , electrode , snail , chromatography , biochemistry , biology , ecology , organic chemistry
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 Ca(2+)‐injecting current. To increase [Ca2+]i by 0.1 microM, the amount of calcium ions injected during this stage had to be 36 +/‐ 20 microM 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 Ca(2+)‐injecting current, which was quite low (corresponding to injection of 0.39 +/‐ 0.20 microM s‐1 for a 0.1 microM change of [Ca2+]i). This may represent the functioning of Ca(2+)‐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 microM s‐1 per cell volume. 5. No activation of Ca(2+)‐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.