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. 1993 Mar;462:47–58. doi: 10.1113/jphysiol.1993.sp019542

Calcium clamp in isolated neurones of the snail Helix pomatia.

P Belan 1, P Kostyuk 1, V Snitsarev 1, A Tepikin 1
PMCID: PMC1175288  PMID: 8392572

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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Selected References

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  1. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  2. Celio M. R. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience. 1990;35(2):375–475. doi: 10.1016/0306-4522(90)90091-h. [DOI] [PubMed] [Google Scholar]
  3. Connor J. A. Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6179–6183. doi: 10.1073/pnas.83.16.6179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fink L. A., Connor J. A., Kaczmarek L. K. Inositol trisphosphate releases intracellularly stored calcium and modulates ion channels in molluscan neurons. J Neurosci. 1988 Jul;8(7):2544–2555. doi: 10.1523/JNEUROSCI.08-07-02544.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  6. Hermann A., Hartung K. Properties of a Ca2+ activated K+ conductance in Helix neurones investigated by intracellular Ca2+ ionophoresis. Pflugers Arch. 1982 May;393(3):248–253. doi: 10.1007/BF00584078. [DOI] [PubMed] [Google Scholar]
  7. Kostyuk P. G., Belan P. V., Tepikin A. V. Free calcium transients and oscillations in nerve cells. Exp Brain Res. 1991;83(2):459–464. doi: 10.1007/BF00231173. [DOI] [PubMed] [Google Scholar]
  8. Meldolesi J., Volpe P., Pozzan T. The intracellular distribution of calcium. Trends Neurosci. 1988 Oct;11(10):449–452. doi: 10.1016/0166-2236(88)90197-x. [DOI] [PubMed] [Google Scholar]
  9. Osipchuk Y. V., Wakui M., Yule D. I., Gallacher D. V., Petersen O. H. Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells. EMBO J. 1990 Mar;9(3):697–704. doi: 10.1002/j.1460-2075.1990.tb08162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Persechini A., Moncrief N. D., Kretsinger R. H. The EF-hand family of calcium-modulated proteins. Trends Neurosci. 1989 Nov;12(11):462–467. doi: 10.1016/0166-2236(89)90097-0. [DOI] [PubMed] [Google Scholar]
  11. Somlyo A. P., Somlyo A. V. Flash photolysis studies of excitation-contraction coupling, regulation, and contraction in smooth muscle. Annu Rev Physiol. 1990;52:857–874. doi: 10.1146/annurev.ph.52.030190.004233. [DOI] [PubMed] [Google Scholar]

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