Until now the only reported effect of depolarization on the intracellular pH (pHi) of excitable cells in an acidification of the cell cytoplasm. It seems unlikely that this could be a direct effect of membrane potential because pHi is known to be regulated by an electroneutral mechanism and in most cells H+ ions are not in equilibrium with the membrane potential (Em). In any case the membrane conductance to H+ ions would be expected to be small because they are at such low concentrations on either side of the cell membrane. But it is possible that the H+ ion permeability of the membrane increases on depolarization just like that of other ions in the bathing medium depolarization just like that of other ions in the bathing medium (Na+, K+ and Ca2+ for example). To test this idea we have made pHi measurements on molluscan neurones under voltage-clamp. Our findings, presented here, provide evidence for a large increase in H+ ion permeability in depolarized cells. We suggest that this increase in proton conductance may be the basis for the "nonspecific' currents previously described in perfused molluscan neurones and we assess the physiological significance of this newly discovered pathway.
SUMMARY1. Intracellular pH (pHi), Cl-and Na+ levels were recorded in snail neurones using ion-sensitive micro-electrodes, and the mechanism of the pH1 recovery from internal acidification investigated.2. Reducing the external HCO3-concentration greatly inhibited the rate of pHi recovery from HC1 injection.3. Reducing external Cl-did not inhibit pH1 recovery, but reducing internal Cl-, by exposing the cell to sulphate Ringer, inhibited pH1 recovery from CO2 application.4. During pHi recovery from C02 application the internal Cl-concentration decreased. The measured fall in internal Cl-concentration averaged about 25 % of the calculated increase in internal HCO3-.5. Removal of external Na inhibited the pH1 recovery from either C02 application or HCl injection.6. During the pHi recovery from acidification there was an increase in the internal Na+ concentration ([Na+]i). The increase was larger than that occurring when the Na pump was inhibited by K-free Ringer.7. The increase in [Na+]i that occurred during pHi recovery from an injection of HC1 was about half of that produced by a similar injection of NaCl.8. The inhibitory effects of Na-free Ringer and of the anion exchange inhibitor SITS on pH1 recovery after HC1 injection were not additive. 9. It is concluded that the pH, regulating system involves tightly linked Cl--HC03-and Na+-H+ exchange, with Na entry down its concentration gradient probably providing the energy to drive the movement inwards of HCO3-and the movement outward of Cl-and H+ ions.
SUMMARY1. The construction and properties of a new design of pH-sensitive micro-electrode are described. The electrodes are very durable, and have a recessed configuration so that only the extreme tip, which can be as small as 1 ,um in diameter, needs to enter the cell.2. The average intracellular pH in thirty-two snail neurones was 7x4. This was not in accord with a passive distribution of H+ ions across the cell membrane.3. Changing membrane potential or external pH had only slow effects on internal pH.4. Removing external K had no effect, and removing external Na had only slow and variable effects on intracellular pH.5. Anoxia, azide and DNP all caused a slow fall in internal pH. 6. External CO2 caused large and rapid decreases in internal pH, which external bicarbonate appeared to offset slowly. Injected bicarbonate increased internal pH.7. The size of the pH changes caused by CO2 suggested a minimum intracellular buffering power of 25 m-equiv H+/unit pH per 1., equivalent to that of 150 mm Tris maleate, pH 7*4. 8. External ammonia caused a large and rapid increase in internal pH, while the injection of ammonium ions had the opposite effect.
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