ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump: evidence for allosteric effects of intracellular ATP and extracellular sodium

Abstract

SUMMARY1. A method has been developed for regenerating [y32P]ATP of constant specific activity within resealed red cell ghosts, and for measuring its hydrolysis. The method may be used to follow the hydrolysis of ATP at concentrations down to 1 /tM, and for periods long enough for the ATP at these very low concentrations to turn over several hundred times.2. Using this method we have been able to show that the 'uncoupled' efflux of Na caused by the Na pump when resealed red cell ghosts are incubated in (Na + K)-free media is associated with a hydrolysis of ATP. The stoicheiometry is roughly 2-3 Na ions expelled per molecule of ATP hydrolysed.3. Measurements of ATP hydrolysis and Na efflux as functions of intracellular ATP concentration have shown that uncoupled Na efflux, and its associated ATP hydrolysis, are saturated at intracellular ATP concentrations in the region of 1 /iM. 4. Measurement of ATP hydrolysis as a function of ATP concentration in resealed ghosts incubated in a K-containing medium gave a complicated activation curve suggesting the involvement of high-affinity (Km ca. 1 /tM) and low-affinity (K,,, ca. 100 /LM) sites.5. When resealed ghosts containing about 1 /lM-ATP were incubated in a Na-free or in a high-Na medium, the addition of K to the medium reduced the rate of ouabain-sensitive ATP hydrolysis.6. Ouabain-sensitive ATP hydrolysis in resealed ghosts incubated in K-free choline media was inhibited by external Na at low concentrations (Ki < 1 mM), but this inhibition was reversed as the external Na concentration was further increased.

Stimulation and Inhibition of the Sodium Pump by Cardioactive Steroids in Relation to Their Binding Sites and Their Inotropic Effect on Guinea‐pig Isolated Atria

“…The study of this action has improved our knowledge of cellular ionic regulation (see Glynn, 1964;Glynn & Karlish, 1976). In guinea-pig atria, our observations summarized in The observation that potassium does affect the apparent affinity of these sites for ouabain is in agreement with others (Baker & Willis, 1970;Brading & Widdicombe, 1974).…”

“…Recent reports have shown that several sites are involved in the action of cations on the Na pump (Cavieres & Ellory. 1975;Glynn & Karlish. 1976).…”

Interaction of external alkali metal ions with the Na-K pump of human erythrocytes: a comparison of their effects on activation of the pump and on the rate of ouabain binding.

“…A possible candidate for this class of sites is the sites from which Na is discharged following translocation outward. Glynn and Karlish (1976) observed an external site with low affinity for Na on Na-K pumps of human red cells at which the binding of Na stimulated activity of Na-ATPase. It is tempting to consider a single external site or set of sites with low affinity for Na, but it is not obvious how binding of Na could stimulate both ouabain binding and Na-ATPase at the same site.…”

“…The inhibition of the pump by Na is presumably by competition with K for the loading sites under these experimental conditions, whereas the promotion of ouabain binding is proposed here to be by some unspecified mechanism at the discharge sites. (Although inhibition of K influx by Na involves competition, there may be other types of inhibitory effects as well [Glynn and Karlish, 1976;Sachs, 1977]. )…”

Effect of Extracellular pH on Presteady-State and Steady-State Current Mediated bythe Na+/K+ Pump

“…4B) show that at pH 7.7 there was a very small component of outward current blocked by H 2 DTG (−0.14 μA cm −2 at Ba). This small H 2 DTG-sensitive outward current and 22 Na + efflux can be explained either by forward pumping, with protons acting as K + congeners (3Na + /2H + exchange) (Polvani & Blostein, 1988), or by 3Na + /ATP (uncoupled) electrogenic transport (Garrahan & Glynn, 1967;Glynn & Karlish, 1976;Cornelius, 1989;1990). The measured 22 Na efflux of 3.8 pmol cm −2 s −1 would result in an outward current of 0.37 μA cm −2 if it were generated by uncoupled 3Na + /ATP transport.…”