The effects of ATP on the interactions between monovalent cations and the sodium pump in dialysed squid axons

Abstract

The efflux of Na in dialyzed axons of the squid was used to monitor the sidedness of the interactions of the Na pump with Na+, K+ and ATP. The axons were under conditions such that most of the Na efflux went through the Na pump by means of a complete cycle of ATP hydrolysis. With 310 mM-K1+, 70 mM-Nai+ and 10 mM-K+ artificial sea water (ASW) 97% of the Na efflux was abolished by removal of ATP. Na efflux was stimulated by ATP with a K1/2 of about 200 .mu.M. This is similar to the K1/2 of 150 .mu.M for the ATP dependence of a ouabain-sensitive Na,K-ATPase activity in membrane fragments isolated from squid optical nerves. A 100-fold reduction in the ATP concentration (from 3-5 mM to 30-50 .mu.M) increased the apparent affinity of the Na pump for Ko+ about 8-fold. The maximal rate of Ko+-stimulated Na efflux was reduced by a similar factor. Analogous results were seen in axons dialyzed with 310 mM-Ki+ or without Ki+. The relative effectiveness of external monovalent cations as activators of the Na efflux was a function of the ATP concentration inside the axon. With 3-5 mM-ATP the order of effectiveness was K+ > NH4+ > Rb+. With 30-50 .mu.M-ATP the sequence was NH4+ > K+ > Rb+. These results were not affected by the removal of Ki+. When the ATP concentration was 3 mM and the Nai+ concentration 70 mM, the removal of Ki+ produced a slight and reversible increase in the total efflux of Na (15%) and no change in the ATP-dependent Na efflux. When the ATP concentration was reduced to 30-50 .mu.M, or the Nai+ concentration lowered to 5-10 mM, the removal of Ki+ reversibly increased the total and the ATP-dependent efflux of Na. The largest increase in Na efflux was seen when both ATP and Nai+ were simulatenously reduced. The ATP-dependent extra Na efflux resulting from the exclusion of Ki+ was abolished by 10-4 M-ouabain in the sea waters. The increase in the ATP-dependent Na efflux obserbed in axons dialyzed with O Ki+ + 10 mM-K+ ASW was not seen in axons perfused with 310 mM-Ki+ + 450 mM- K+ ASW. Both experimental conditions gave rise to a similar (and small) ATP-independent and ouabain-insensitive efflux of Na. The effects of removing Ki+ on the Na pump are probably not due to the simultaneous membrane depolarization. Ki+ apparently has an inhibitory effect on the Na pump, and that effect is antagonized by Nai+ and ATP. Results are consistent with the idea that the same conformation of the Na pump (and Na,K-ATPase) can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. This enzyme conformation produces an enzyme-K complex from which K+ are not easily released unless high concentrations of ATP are present. This stresses a non-phosphorylating regulatory role of ATP.