Phosphate from the phosphointermediate (EP) of the human red blood cell Na/K pump is coeffluxed with Na, in the absence of external K

This study is concerned with Na/K pump-mediated phosphate efflux that occurs during uncoupled Na efflux in human red blood cells. Uncoupled Na efflux is known to be a ouabain-sensitive mode of the Na/K pump that occurs in the absence of external Nao and Ko. Because this efflux (measured with 22Na) is also inhibited by 5 mM Nao, the efflux can be separated into a Nao-sensitive and a Nao-insensitive component. Previous work established that the Nao-sensitive efflux is actually comprised of an electroneutral coefflux of Na with cellular anions, such as SO4 (as 35SO4). The present work focuses on the Nao-insensitive component in which the principal finding is that orthophosphate (P(i)) is coeffluxed with Na in a ouabain-sensitive manner. This P(i) efflux can be seen to occur, in the absence of Ko, in both DIDS-treated intact cells and resealed red cell ghosts. This efflux of P(i) was shown to be derived directly from the pump's substrate, ATP, by the use of resealed ghosts made to contain both ATP and P(i) in which either the ATP or the P(i) were labeled with, respectively, [gamma-32P]ATP or [32P]H3PO4. (These resealed ghosts also contained Na, Mg, P(i), SO4, Ap5A, as well as an arginine kinase/creatine kinase nucleotide regenerating system for the control of ATP and ADP concentrations, and were suspended usually in (NMG)2SO4 at pH 7.4.) It was found that 32P was only coeffluxed with Na when the 32P was contained in [gamma-32P]ATP and not in [32P]H3PO4. This result implies that the 32P that is released comes from ATP via the pump's phosphointermediate (EP) without commingling with the cellular pool of P(i). Ko (as K2SO4) inhibits this 32P efflux as well as the Nao-sensitive 35SO4 efflux, with a K0.5 of 0.3-0.4 mM. The K0.5 for inhibition of P(i) efflux by Ko is not influenced by Nao, nor can Nao act as a congenor for Ko in any of the flux reactions involving Ko. The stoichiometry of Na to SO4 and Na to P(i) efflux is approximately 2:1 under circumstances where the stoichiometry of Na effluxed to ATP utilized is 3:1. From these and other results reported, it is suggested that there are two types of uncoupled Na efflux that differ from each other on the basis of their sensitivity to Nao, the source (cellular vs substrate) and kind of anion (SO4 vs P(i)) transported.(ABSTRACT TRUNCATED AT 400 WORDS)