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División de Biofísica, Instituto de Investigación Medica M.y M. Ferreyra, Casilla de Correo 389, 5000 Córdoba, Argentina

Centro de Biofísica y Bioquímica, I.V.I.C., Apartado 1827, Caracas, Venezuela

The Marine Biological Laboratory, Woods Hole, Mass., U.S.A.

1. The efflux of Na in dialysed axons of the squid has been used to monitor the sidedness of the interactions of the Na pump with Na⁺ ions, K⁺ ions 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.

2. With 310 mM-K_i⁺, 70 mM-Na_i⁺ and 10 mM-K⁺ artificial sea water (ASW) more than 97% of the Na efflux was abolished by removal of ATP. The efflux of Na was stimulated by ATP with a K of about 200 µM. This is similar to the K of 150 µM found for the ATP dependence of a ouabain-sensitive Na,K-ATPase activity in membrane fragments isolated from squid optical nerves.

3. A 100-fold reduction in the ATP concentration (from 3-5 mM to 30-50 µM) increased the apparent affinity of the Na pump for K_o⁺ about 8-fold. In addition, the maximal rate of K_o⁺-stimulated Na efflux was reduced by a similar factor. Analogous results were seen in axons dialysed with 310 mM-K_i⁺ or without K_i⁺.

4. 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 µM-ATP the sequence was NH₄⁺ » K⁺ » Rb⁺. These results were not affected by the removal of K_i⁺.

5. When the ATP concentration was 3 mM and the Na_i⁺ concentration 70 mM, the removal of K_i⁺ 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 µM, or the Na_i⁺ concentration lowered to 5-10 mM, the removal of K_i⁺ reversibly increased the total and the ATP-dependent efflux of Na. The largest increase in Na efflux was seen when both ATP and Na_i⁺ were simultaneously reduced. The ATP-dependent extra Na efflux resulting from the exclusion of K_i⁺ was abolished by 10^-4 M-ouabain in the sea waters.

6. The increase in the ATP-dependent Na efflux observed in axons dialysed with 0 K_i⁺ + 10 mM-K⁺ ASW was not seen in axons perfused with 310 mM-K_i⁺ + 450 mM-K⁺ ASW. However, both experimental conditions gave rise to a similar (and small) ATP-independent and ouabain-insensitive efflux of Na. This indicates that the effects on the Na pump of removing K_i⁺ are not due to the simultaneous membrane depolarization. In addition, it suggests that K_i⁺ has an inhibitory effect on the Na pump, and that that effect is antagonized by Na_i⁺ and ATP.

7. The present 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⁺ ions are not easily released unless high concentrations of ATP are present. This also stresses a non-phosphorylating regulatory role of ATP.