THE ALPHA-1 NA-K+ PUMP OF THE DAHL SALT-SENSITIVE RAT EXHIBITS ALTERED NA+ MODULATION OF K+ TRANSPORT IN RED-BLOOD-CELLS()

The properties of the al Na+-K+ pump were compared in Dahl salt-sensit ive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitiv e fluxes (mmol/liter cell x hr = FU, Mean +/- SE) in red blood cells ( RBCs) and varying internal and external (o) Na+ and K+ concentrations. Kinetic parameters of several modes of operation, i.e., Na+/K+, K+/K, Na+/Na+ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n = 12 rats of each strain) into fresh cells incubated in 140 mm Na+ 5 mm K+, ouabain-sensitive K- influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na+ efflux and Na(i) were similar in both strains. Thus, the coupling ratio between unidirection al Na+ : K+ fluxes was significantly higher in DS than in DR cells at similar RBC Na+ content. In the presence of 140 mm Na(o), activation o f ouabain-sensitive K+ influx by K(o) had a lower K(m) and V(max) in D S as estimated by the Garay equation (N = 2.70 +/- 0.33, K(m) 0.74 +/- 0.09 mm; V(max) 2.87 +/- 0.09 FU) than in DR rats (N = 1.23 +/- 0.36, K(m)2.31 +/- 0.16 mM; V(max) 5.70 +/- 0.52 FU). However, the two kine tic parameters were similar following Na(o) removal. The activation of ouabain-sensitive K+ influx by Na(i) had significantly lower V(max) i n DS (9.3 +/- 0.4 FU) than in DR (14.5 +/- 0.6 FU) RBCs but similar K( m). These data suggest that the low K+ influx in DS cells is caused by a defect in modulation by Na(o) and Na(i). Na+ efflux showed no diffe rences in Na(i) activation or trans effects by Na(o) and K(o), thus ac counting for the different Na+ : K+ coupling ratio in the Dahl strains . Further evidence for the differences in the coupling of ouabain-sens itive fluxes was found in studies of net Na+ and K+ fluxes, where the net ouabain-sensitive Na+ losses showed similar magnitudes in the two Dahl strains while the net ouabain-sensitive K+ gains were significant ly greater in the DR than the DS RBCs. Ouabain-sensitive Na+ influx an d K+ efflux were also measured in these rat RBCs. The inhibition of ou abain-sensitive Na+ influx by K(o) was fully competitive for the DS bu t not for the DR pumps. Thus, for DR pumps, K(o) could activate higher K+ influx in DR pumps without a complete inhibition of ouabain-sensit ive Na+ influx. This behavior is consistent with K(o) interaction with distinct Na+ and K+ transport sites. In addition, the inhibition of K + efflux by Na(i) was different between Dahl strains. Ouabain-sensitiv e K+ efflux at Na(i) level of 4.6 mmol/liter cell, was significantly h igher in DS (3.86 +/- 0.67 FU) than in DR (0.86 +/- 0.14 FU) due to a threefold higher K50 for Na(i)-inhibition (9.66 +/- 0.41 vs. 3.09 +/- 0.11 mmol/liter cell. This finding indicates that Na+ modulation of K transport is altered at both sides of the membrane. The dissociation of Na+ modulatory sites of K+ transport from Na+ transport sites obser ved in RBCs of Dahl strains suggests that K+ transport by the Na+-K+ p ump is controlled by Na+ allosteric sites different from the Na+ trans port sites. The alterations in K+ transport may be related to the amin o acid substitution (Leu/Gln276) reported for the cDNA of the al subun it of the Na+-K+ pump in the DS strain or to post-translational modifi cations during RBC maturation.