Calcium clamp in isolated neurones of the snail Helix pomatia.

P. Belan, P. Kostyuk, V. Snitsarev, A. Tepikin

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)47-58
Number of pages12
JournalThe Journal of Physiology
Volume462
Issue number1
DOIs
StatePublished - 1 Mar 1993

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Helix (Snails)
Microelectrodes
Calcium
Neurons
Injections
Ions
Cell Size
Calcium Channels
Constriction
Membrane Potentials
Cytosol
Potassium
Buffers
Electrodes
Cytoplasm
Membranes

Cite this

Belan, P. ; Kostyuk, P. ; Snitsarev, V. ; Tepikin, A. / Calcium clamp in isolated neurones of the snail Helix pomatia. In: The Journal of Physiology. 1993 ; Vol. 462, No. 1. pp. 47-58.
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abstract = "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.",
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Calcium clamp in isolated neurones of the snail Helix pomatia. / Belan, P.; Kostyuk, P.; Snitsarev, V.; Tepikin, A.

In: The Journal of Physiology, Vol. 462, No. 1, 01.03.1993, p. 47-58.

Research output: Contribution to journalArticle

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T1 - Calcium clamp in isolated neurones of the snail Helix pomatia.

AU - Belan, P.

AU - Kostyuk, P.

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N2 - 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.

AB - 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.

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