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. 1983 Jan;334:189–212. doi: 10.1113/jphysiol.1983.sp014489

The effects of injection of calcium ions and calcium chelators on calcium channel inactivation in Helix neurones.

T D Plant, N B Standen, T A Ward
PMCID: PMC1197309  PMID: 6306229

Abstract

1. The effects of intracellular injection of Ca, EGTA and EGTA/Ca buffers on inward currents flowing through the Ca channel in Helix aspersa neurones were studied under voltage clamp. 2. Inward currents were reversibly reduced by Ca injection. The extent of the reduction was dependent on the size and duration of the injection. Recovery from injection was exponential with a time constant around 18 s at 18-20 degrees C. 3. In our salines, which contained tetraethylammonium chloride and 4-aminopyridine, no outward current was activated by Ca injection at the holding potential. A given Ca injection reduced the inward current by the same fraction in 25 mM- and 2 . 5 mM-Sr and also at different test potentials. We conclude that Ca injection does not activate an outward current. 4. Mean resting ionized internal Ca concentration, [Ca]i, measured with a Ca-sensitive electrode was 1.9 X 10(-7) M. Our injections increased this by less than 10(-5) M, as expected if most of the injected Ca is bound. Constant-field or Eyring rate theory considerations suggest that this rise in [Ca]i would not significantly affect the inward current through open Ca channels and we conclude, therefore, that Ca injection causes Ca channel inactivation. 5. The effect of Ca injection was blocked by prior injection of EGTA. Extracellular application of carbonyl cyanide m-chlorophenylhydrazone increased the effect of Ca injection. 6. Ca injection does not inactivate Ca channels by lowering pHi since large H+ injections only caused small irreversible decreases in inward current. 7. EGTA injection increased peak Ca current (ICa) by around 30% and decreased the rate and extent of inactivation. Some inactivation remained, however, even after maximal EGTA injections. 8. Injection of EGTA/Ca buffers with free [Ca] less than 1.8 X 10(-7) M increased peak ICa, while buffers with free [Ca] greater than 8.9 X 10(-7) M decreased ICa. 9. Our results support the suggestion that Ca ions cause Ca inactivation by binding to a site which is accessible from the inside of the cell, and also suggest that there is some steady-state inactivation present at physiological [Ca]i. A simple model is presented which describes the decline of Ca current in terms of Ca binding to a site accessible from the cytoplasm.

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Selected References

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