TISSUE-SPECIFIC AND ISOFORM-SPECIFIC KINETIC-BEHAVIOR OF THE NA,K-ATPASE

The objective of this study has been to delineate the side-specific ef fects of Na+ and K+ on the transport kinetics of tissue-specific Na/K pumps. Two experimental systems have been used. In one, Na/K pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were deli vered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individ ually transfected into HeLa cells as in previous studies (Jewell, E. A ., and Lingrel, J.B (1991) J. Biol. Chem. 266, 16925-16930), with the alpha(2) and alpha(3) isoforms rendered relatively resistant to ouabai n by site-directed mutagenesis. Whereas the kidney microsomes comprise the alpha(1) catalytic isoform, the axolemma microsomes were predomin antly alpha(3) (approximate to 60%) with lesser amounts of alpha(2) (a pproximate to 25%) and alpha(1) (approximate to 15%) as measured by th e ouabain-sensitive profile of phosphoenzyme as well as by immunoblott ing with isoform-specific antibodies using membranes of known specific activity as standards (alpha(1) of kidney, alpha(1) and alpha(2) of m uscle). Both systems were analyzed with respect to the effects of vary ing concentrations of cytoplasmic Na+ and extracellular K+ on pump-med iated Rb-86(+)(K+) influx. With the individual isoform-transfected HeL a cells and monensin added to vary and control the intracellular Na+ c oncentration, differences in apparent affinities of the alpha(3) isofo rm compared with the alpha(1) and alpha(2) isoforms were observed, i.e . a approximate to 3-fold higher affinity for extracellular K+ and app roximate to 4-fold lower affinity for cytoplasmic Na+. Thus, in the pr esence of 10 mM extracellular Na+, apparent K-0.5 values for extracell ular K+ activation of K+(Rb+) influxes were 0.22 +/- 0.02 mM for alpha (1), 0.20 +/- 0.02 mM for alpha(2), and 0.09 +/- 0.01 mM for alpha(3). At high intracellular K+ (greater than or equal to 100 mM) and satura ting extracellular K+ concentrations, apparent K-0.5 values for cytopl asmic Na+ activation were 17.6 +/- 1.1 mM for alpha(1), 19.7 +/- 1.0 m M for alpha(2), and 63.5 +/- 9.1 mM for alpha(3). The functional diffe rences observed with the individual isoform-transfected cells were com pletely consistent with the kinetic differences observed with the axol emma and kidney pumps fused into erythrocytes. Axolemma pumps had a ap proximate to 3-fold lower K-0.5 for extracellular K+ and a approximate to 2-fold higher K-0.5 for cytoplasmic Na+. In the HeLa transfectants , differences in affinities for cytoplasmic Na+ were associated with d ifferences in the steady-state intracellular Na+ concentration, i.e. 2 7.5 mM in alpha(3)-transfected cells compared with 15.7 and 19.7 mM in alpha(1)- and alpha(2)-transfected cells, respectively.