ATP as a modulator of Rb+ occlusion by Na+/K+-ATPase through the direct route

SPIAGGI AJ; GONZÁLEZ-LEBRERO RM; GARRAHAN PJ; ROSSI RC

Pacific Grove, CA

Congreso; 13th International P-type ATPase meeting; 2011

ASMBM

Besides its phosphorylating function on Na+/K+–ATPase, ATP accelerates K+ deocclusion and seems necessary to direct the release of occluded K+ toward the intracellular face of the enzyme. Interestingly, studies on the equilibrium between free and occluded Rb+ (a K+ analog) obtained through the direct route showed that, even in the presence of ATP, the proportion of states with bound but not occluded Rb+ was negligible.We used an unsided, purified preparation of pig-kidney Na+/K+–ATPase and a rapid-quenching and filtration method to test if acceleration of Rb+ deocclusion by ATP is accompanied by an increase in the rate of occlusion (vocc), thus making the maximal amount of occluded Rb+ independent of [ATP]. The literature shows evidence both in favor and against this possibility.Our results show that:i. When [ATP] is increased at constant [Rb+], vocc decreases along a rectangular hyperbola whose K0.5 is independent of [Rb+] and comparable to the dissociation constant of the nucleotide from the catalytic site.ii. The relative change in vocc decreases toward zero as [Rb+] raises, meaning that the process that leads to Rb+ occlusion occurs at the same rate in both the free and the ATP-bound enzyme when the Rb+-binding sites are fully loaded.iii. At saturating [ATP], and up to 500 μM Rb+, vocc raises along a curve that appears to be the beginning of a sigmoid function of [Rb+] with initial slope greater than zero.These results suggest that neither the acceleration of K+ deocclusion nor the inhibition of K+ occlusion exerted by ATP are strong enough to prevent that species with bound K+ are mostly in the occluded form.