The sidedness of the direct route of occlusion of K+ in the Na+/K+-ATPase

R. M. GONZÁLEZ-LEBRERO; S. B. KAUFMAN; P. J. GARRAHAN; R. C. ROSSI

ANN. N. Y. ACADEMY SCIEN.

NYAS

Año: 2003 vol. 986 p. 301 - 303

1749-6632

Occlusion of up to two K+ (or its congeners) per Na+/K+–ATPase molecule takes place either in media lacking Na+, Mg2+ and ATP (direct route) or during ATPase activity, after K+ has promoted dephosphorylation of the enzyme (physiological route). In the latter condition, K+ occlusion takes place through the extracellular sites of the enzyme. It is usually assumed that the direct route leads to K+ occlusion through intracellular sites. However, there is no direct evidence to support this claim and some experimental results seem to disagree with this view. We have reported results indicating that the direct route can lead to the sequential occlusion of one or two Rb+ according to a single-file mechanism. Similar mechanisms were proposed elsewhere to explain results obtained in the presence of MgPi. The sequential occlusion of the two Rb+ can be envisioned as taking place into a narrow channel with gated entrances, one to each side of the membrane as shown in the following scheme:                      ( )E( ) + Rb <-> (Rb)E( ) <-> ( )E(Rb) + Rb <-> (Rb)E(Rb) Occlusion occurs only when both gates are closed. We assume that, under the conditions of our experiments, one of the gates (that in position I, represented at the left side of E) opens much more frequently than the other so that almost all the exchange of Rb+ will take place through this gate. For Rb+ to become occluded, it must bind to a site in position I and jump to a deeper position, II, before a second Rb+ can be bound and occluded. Release of Rb+ follows the reverse pathway. Using the assumption that the binding of MgPi to the enzyme promotes deocclusion toward the extracellular gate to assign a sidedness to positions I and II, we here provide experimental evidence to answer whether the occlusion of K+ in the Na+/K+-ATPase via the direct route takes place through intracellular or extracellular sites of the enzyme.