NA+-DEPENDENT CL-HCO3 EXCHANGE IN THE SQUID AXON - DEPENDENCE ON EXTRACELLULAR PH

Intracellular pH (pH(i)) in squid giant axons recovers from acid loads by means of a Na+-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pH(o)) in the range 7.1-8.6. We altered pH(o) in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO-]o and [NaCO3-]o. Axons (initial pH(i) approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pH(i), measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (J(H)) was computed from the rate of pH(i) recovery (i.e., increase) in the presence of Na+ and HCO3-. When pH(o) was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-], values, J(H) increased with pH(o) in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, J(H) also increased with pH(o), though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, J(H) was insensitive to pH(o) changes. Finally, with method (iv), which involved changes in neither [HCO3-]o nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, J(H) also was insensitive to pH(o) changes. We found that decreasing pH(o) from 8.6 to 7.1 caused the apparent K(m) for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas J(max) was relatively stable. Decreasing pH(o) from 8.6 to 7.4 caused the apparent K(m) values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas J(max) was relatively stable. These data are consistent with the ion pair model, and severely restrict kinetic parameters for other models.