Proton Block of Rat Brain Sodium Channels

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 a t tempt to minimize interpretat ional ambiguities, the experiments were done in 1.0 M NaCI (buffered to the appropr ia te 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 ~ 4.9. As the p H was decreased below 6.5, the single-channel current-vol tage (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 p H of the other solution being held constant at 7.4), the titration behavior was different for extraand intracellular acidification. With extracellular acidification, the reduction in g could still be approximated by a simple titration function with a pK of ~ 4.6, and there was a pronounced rectification at pHs < 6 (cf. Woodhull, A. M. 1973. Journal of General Physwlogy. 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 ~4 .6 at an apparent electrical distance of ~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 vol tage-dependent block could be approximated by assuming that protons enter the pore and bind to a site with a pK of ~ 4.1 at an apparent electrical distance of ~ 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. Address reprint requests to Dr. Olaf S. Andersen, Department of Physiology and Biophysics, Cornell University Medical College, 1300 York Avenue, New York, NY 10021-4896. J. GEN. PHYSIOL. © The Rockefeller University Press • 0022-1295/93/01/0027/17 $2.00 Volume 101 January 1993 27-43 27 on A ril 3, 2017 D ow nladed fom Published January 1, 1993