Voltage‐dependent intracellular pH in Helix aspersa neurones.

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Abstract
1. The intracellular pH (pHi) of large nerve cells from the mollusc, Helix aspersa, was measured with pH-sensitive micro-electrodes. Cells were held under voltage clamp and the effect on pHi of different holding potentials was determined. 2. Depolarization of the cell from the resting potential (about -50 mV) to -10 mV produced a fall in pHi that could be reduced by bathing the cell in nominally Ca2+-free saline. 3. At positive holding potentials pHi increased to a steady level that depended upon the electrochemical gradient for H+ across the cell membrane; it shifted by about 1 unit when the external pH was increased from 7 to 8 (or when the membrane potential increased by 58 mV, Thomas and Meech, 1982). 4. The depolarization-induced increase in H+ permeability was insensitive to SITS (4-acetamido-4''-isothiocyanostilbene-2,2''-disulphonic acid, 20 .mu.M), which blocks pHi regulation at the resting potential in these cells (Thomas, 1976). When pHi was displaced from a steady level by ionophoretic injection of HCl, there was a rapid recovery at depolarized potentials even in the presence of SITS. The H+ pathway appeared to be little affected by prolonged periods at positive membrane potentials. 5. The depolarization-induced H+ efflux was insensitive to the metabolic inhibitor CCmP (carbonyl cyanide-m-chlorophenylhydrazone, 20 .mu.M) and persisted in cells bathed in pH-buffered n-methyl glucamine-gluconate. It was also insensitive to DCCD (N, N''-dicyclohexylcarbodiimide, 10-100 .mu.M) and oligomycin (2-10 .mu.g/ml). 6. The H+ pathway could be fully blocked by 1 mM-ZnCl2, 1 mM-LaCl3, 1 mM-CuCl2, 2 mM-CdCl2 or 10 mM-CoCl2. Other divalent ions such as BaCl2 (10 mM) produced a block at membrane potentials near 0 mV but the block was released at more positive potentials. Low levels of LaCl3 (0.1 mM), the organic Ca2+ channel antagonist D600 (100 mg/ml) and high levels of the K+ channel blocker TEA (50 mM) all had similar effects to Ba2+. 7. The K+ channel blocker 4-aminopyridine (10 mM), which blocks H+ currents in perfused Lymnaea neurones (Byerly, Meech and Moody, 1984), has a complex action. It is a weak base and it increases both pHi and the buffering power of the cell cytoplasm. 8. Our main conclusions are that the voltage-activated H+ pathways in Helix is not an ATP-dependent pump, it is not a DCCD-inhibitable proton ATPase and the H+efflux is not coupled to the movement of any other ion. It is inhibited by agents that block Ca2+ channels and it resembles the H+ pathway studied in perfused Lymnaea neurones (Byerly et al. 1984). 9. Just as in Lymnaea, the conductance of the H+ pathway appeared to increase when pHi was reduced. The transmembrane H+ flux is increased by depolarization but may be limited by diffusion-dependent processes in the cell cytoplasm. 10. The H+ pathway may function to maintain pHi near its resting level despite the significant acid load caused by intracellular Ca2+/H+ exchange following Ca2+ influx during trains of action potentials.