Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant axons

The ion transport system responsible for intracellular pH (pHi) regulation in squid giant axons was examined in experiments with pH-sensitive microelectrodes and isotopic fluxes of Na+ and Cl-. In one study, axons were acid-loaded and the rate of the subsequent pHi recovery was used to calculate the acid extrusion rate. There was an absolute dependence of acid extrusion on external Na+, external HCO-3 (at constant pH), and internal Cl-. Furthermore, the dependence of the acid extrusion rate on each of these three parameters was described by Michaelis-Menten kinetics. Acid extrusion was stimulated by an acid pHi, required internal ATP, and was blocked by external 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). Under a standard set of conditions (i.e., [HCO-3]o = 12 mM, pHo = 8.00, [Na+]o = 425 mM, [Cl-]i = 150 mM, [ATP]i = 4 mM, pHi = 6.5, and 16 degrees C), the mean acid extrusion rate was 7.5 pmol X cm-2 X s-1. In a second study under the above standard conditions, the unidirectional Na+ efflux (measured with 22Na) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean influx was about 3.4 pmol X cm-2 X s-1. This net influx required external HCO-3, internal Cl-, and acid pHi, internal ATP, and was blocked by SITS. In the final series of experiments under the above standard conditions, the unidirectional Cl- influx (measured with 36Cl) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean efflux was approximately 3.9 pmol X cm-2 X s-1. This net efflux required external HCO-3, external Na+, an acid pHi, internal ATP, and was blocked by SITS. We conclude that the pHi-regulating system mediates the obligate net influx of HCO-3 (or equivalent species) and Na+ and the net efflux of Cl- in the stoichiometry of 2:1:1. The transport system is stimulated by intracellular acid loads, requires ATP, and is blocked by SITS.