Rapid release of 45Ca from an occluded state of the Na,K-pump

45Ca is bound to the occluded state of the Na,K-pump, apparently at K+ sites. Only one 45Ca ion is bound in place of two K+ ions, with an affinity approximately 0.08 mM; K+ competes with an apparent affinity approximately 0.04 mM. 45Ca is released rapidly from Na,K-ATPase in the presence of ATP or ADP, presumably to the intracellular medium. The rate constant of 45Ca release with ATP is greater than 100 s-1 at 20 degrees C, more than twice as fast as the rate of release of 42K from the occluded state. Phosphorylation of Na,K-ATPase with MgPi, which would lead to release of occluded K+ or Rb+ to the extracellular face of the membrane, stabilizes occluded 45Ca. 45Ca release is slower immediately after exposure to MgPi than after a rinse in the absence of Pi indicating that in the former circumstance the rate of 45Ca release is limited by dephosphorylation; 45Ca release is even slower after exposure to Mg2+ arsenate, consistent with dearsenylation being slower than dephosphorylation. When limited by dephosphorylation, the rate of 45Ca release is dependent on the species of monovalent cation present, increasing in the order N-methylglucamine less than Cs+ less than Li+ less than Na+ less than Rb+ less than K+. When the 45Ca occluded state is exposed to K + Mg + Pi and then to Na+ + Mg2+ + ATP, the exposure to K+ is "remembered," indicating simultaneous occlusion of 45Ca and K+. The apparent affinity for K+ in formation of this state is 10-50 mM, and the rate of release of K+ is approximately 2 s-1. Ca2+ has effects on the release of 86Rb from the occluded state: With ATP, Ca2+ acts like Mg2+ by stimulating 86Rb release at low concentrations and inhibiting at high concentrations; with MgPi, Ca2+ inhibits 86Rb release, presumably by preventing phosphorylation. Thus, Ca2+ has two actions on the Na,K-pump as studied here: one as a Mg2+ congener, and another as a K+ congener at transport sites. In the latter role Ca2+ is unusual in that it appears to be able to bind to the transport sites from the intracellular face of the pump and to become occluded, but unable to be released from extracellular sites.