In the range of pH examined (5.2-10), variations of internal pH from high to low values result in a reversible decrease of the conductance of the open K channels, without significantly affecting the kinetics parameters. A linear plot of the conductance versus internal pH suggests the existence of a titratable group that has an apparent pKa of about 6.9, and that is accessible to protons only from the intracellular side of the membrane.

Sodium channels in plasma membranes can be blocked by a large variety of toxins and local anaesthetics. This property, however, is not confined to relatively large molecules. For instance, extracellularly applied small ions like hydrogen may also prevent the passive transport of permeant cations across open Na+ channels. A typical feature of this phenomenon is that the blocking action of hydrogen is gradually relieved by increasing the voltage applied across the membrane. Although in the frog skeletal muscle there is no clear evidence for a similar intracellular action, we report here for the squid giant axon remarkable effects on the ionic permeability of Na+ channels when the internal perfusate contains an excess of protons. Analysing the action of low pH inside and outside the fibre in terms of a kinetic model, we could conclude that Na+ channels in squid axons are controlled by two independent groups: one with an apparent pKa of 4.6 and the other with pKa 5.8, the former feeling one-fifth of the applied membrane potential, the latter three-quarters. As with pharmacological agents, we also show that the voltage-dependence of the H+ blockage is not affected by the presence of the inactivation gate.

Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution.

Squid giant axons were used to study the reversible effects of high intracellular pH (pHi) on gating currents. Under depolarization, when Na channels are activated, internal solutions buffered at high pHi (10.2) affect considerably the time course of gating charge associated with channel closing, QOFF, with almost no alteration of QON records. In particular, at pHi 10.2 the charge corresponding to the fast phase of IgOFF, measured after long depolarizing pulses (7.7 ms), was consistently larger than that recorded at physiological pHi (7.2). This suggests that high pH prevents immobilization of gating charges induced by Na inactivation. In this respect, the present data agree reasonably well with previous observations, which show that pHi greater than 7.2 reversibly removes the fast Na inactivation with little effects on activation kinetics (Carbone, E., P. L. Testa, and E. Wanke, 1981, Biophys. J., 35:393-413; Brodwick, M.S., and D. C. Eaton, 1978, Science [Wash. DC], 200:1494-1496). Unexpectedly, high pH increases the amount of charge associated with the slow phase of IgOFF. In our opinion, this might be the result of either an increment of the net charge produced by the exposure to high pHi or that gating charges that return to the closed state might experience a larger fraction of the potential drop across the membrane (Neumcke, B., W. Schwarz, and R. Stampfli, 1980, Biophys. J., 31:325-332).

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