Effect of measured calcium chloride injections on the membrane potential and internal pH of snail neurones

1. Ion-sensitive micro-electrodes were used to measure changes in intracellular pH (pHi) and internal chloride which resulted from the pressure injection of calcium chloride into identified Helix aspersa neurones. The internal chloride measurement allowed the quantity of calcium chloride injected to be estimated. 2. Application of the metabolic inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCmP) to a calcium-loaded cell caused an increase in the membrane potential comparable to the effect of injecting calcium itself. The effect was not observed in normal cells. 3. When injected in the presence of CCmp, calcium caused a much larger and longer-lasting effect on the membrane potential than that observed in untreated cells. 4. The injection of ruthenium red can increase and/or prolong the hyperpolarization caused by a given quantity of injected calcium. The pHi changes following calcium injection were biphasic and slower than normal. 5. In seven experiments, both hydrogen chloride and calcium chloride were injected into the same cell. The relative changes in pHi corresponded to the production of one hydrogen ion for each calcium ion injected. 6. The relationship between the quantity of calcium injected and the size of the induced hyperpolarization suggested that at least three calcium ions acting cooperatively are required to activate a potassium 'channel'. 7. Injection of barium chloride hyperpolarized the membrane after a delay of about 1 min. After several injections of barium the cell lost this response while retaining its normal response to calcium injection. Injection of barium also caused a slowly developing (biphasic) fall in pHi. 8. We conclude that injected calcium is normally rapidly taken up by mitochondria in exchange for hydrogen ions. If this uptake process is blocked by ruthenium red or CCmP, the calcium is taken up by a second, slower, process which also releases hydrogen ions. When pre-loaded, but not otherwise, the mitochondria will release calcium ions on treatment with CCmP. Injection of barium does not directly affect the membrane conductance, but causes the release of calcium from intracellular binding sites.