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

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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.