title: The effects of ATP on the interactions between monovalent cations and the sodium pump in dialysed squid axons.
abstract: 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((1/2)) of about 200 mum. This is similar to the K((1/2)) of 150 mum 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 mum) 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(+) > NH(4) (+) > Rb(+). With 30-50 mum-ATP the sequence was NH(4) (+) >> 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 mum, 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.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D002414:Cations, Monovalent / Q000494:pharmacology; D049832:Decapodiformes / Q000378:metabolism; D003956:Dialysis; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D002414:Cations, Monovalent; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium