The acid titration function of bilayer-incorporated batrachotoxin (BTX)-modified sodium channels was examined in experiments in which the pH was decreased symmetrically, on both sides of the membrane, or asymmetrically, on only one side. In an attempt to minimize interpretational ambiguities, the experiments were done in 1.0 M NaCl (buffered to the appropriate pH) with channels incorporated into net neutral bilayers. When the pH was decreased symmetrically (from 7.4 to 4.5), the small-signal conductance (g) decreased in accordance with the predictions of a simple (single-site) titration function with a pK of approximately 4.9. As the pH was decreased below 6.5, the single-channel current-voltage (i-V) relation became increasingly rectifying, with the inward current being decreased more than the outward current. When the pH was decreased asymmetrically (with the pH of the other solution being held constant at 7.4), the titration behavior was different for extra- and intracellular acidification. With extracellular acidification, the reduction in g could still be approximated by a simple titration function with a pK of approximately 4.6, and there was a pronounced rectification at pHs less-than-or-equal-to 6 (cf. Woodhull, A. M. 1973. Journal of General Physiology. 61:687-708). The voltage dependence of the block could be described by assuming that protons enter the pore and bind to a site with a pK of approximately 4.6 at an apparent electrical distance of approximately 0.1 from the extracellular entrance. With intracellular acidification there was only a slight reduction in g, and the g-pH relation could not be approximated by a simple titration curve, suggesting that protons can bind to several sites. The i-V relations were still rectifying, and the voltage-dependent block could be approximated by assuming that protons enter the pore and bind to a site with a pK of approximately 4.1 at an apparent electrical distance of approximately 0.2 from the intracellular entrance. Based on the difference between the three g-pH relations, we conclude that there are at least two proton binding sites in the pore and that they can be occupied simultaneously.
