Mechanistic Basis for Kinetic Differences between the Rat α1, α2, and α3 Isoforms of the Na,K-ATPase

Abstract

Previous studies showed that the α1, α2, and α3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na+, and K+. For the rat isoforms transfected into HeLa cells, K'ATP for ATP binding at its low affinity site is lower for α2 and α3 compared with α1; relative to α1 and α2, α3 has a higher K′Na and lower K′K (Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B. and Blostein, R. (1994) J. Biol. Chem. 269, 16668-16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that α2 differs from α1 primarily by a shift in the E1 ⇌ E2 equilibrium in favor of E1 form(s) as evidenced by (i) a -20-fold increase in IC50 for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'ATP compared with α1, the E1 ⇌ E 2 poise of α3 is not shifted toward E1. Distinct intrinsic interactions with Na+ ions are underscored by the marked selectivity for Na+ over Li+ of α3 compared with either α1 or α2 and higher K′Na for cytoplasmic Na+, which persists over a 100-fold range in proton concentration, independent of the presence of K+. The kinetic analysis also suggests α3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na + and K+.