Molecular Mechanism by which Two Lysine Substitutions Alter Na/K-Pump Stoichiometry to Confer High-Salinity Adaptation in Brine Shrimp

Abstract

In most animal cells, the Na/K pump maintains a large electrochemical gradient for Na+ across the plasma membrane by extruding 3 Na+ and importing 2 K+ per ATP molecule hydrolyzed. When adapted to high salinity, brine shrimp (Artemia franciscana) express a pump that has two asparagine-to-lysine substitutions within the normally conserved ion-binding region. To address the molecular mechanisms by which these substitutions may be advantageous, we used two-electrode voltage clamp to evaluate the functional effects of the equivalent substitutions (N333K and N785K) in the Xenopus Na/K pump α subunit. Compared to wild-type, the apparent affinity for K+-activation of pump current without Na+o was reduced 10-fold by N785K, but nearly unaffected by N333K or N333K/N785K. In contrast, both individual mutants displaced the center of the charge-voltage curve measured with Na+o, without K+o, by −80 mV, reflecting a 16-fold decrease in Na+o affinity (20 mV shift per 2-fold change), whereas N333K/N785K induced only a ∼ -40 mV shift. Molecular dynamic simulations (3.3 μs) of these mutants based on the available crystal structures provide an explanation of the effects of individual and concurrent mutations and how these residues are coupled. Simulations of N333K/N785K with a single Na+ or K+ removed from the structures restored ion-binding site stability, consistent with a previous proposal of altered stoichiometry (Jorgensen and Amat, 2008, J. Memb. Biol. 221: 39-49, 2008). To directly assess stoichiometry, we measured 86Rb+ (a K+ congener) uptake in voltage-clamped oocytes and found that, for every charge extruded, wild-type pumps imported 2.11 ± 0.07 (n=40) Rb+, as expected, while N333K/N785K imported 1.01 ± 0.05 (n=13) Rb+. This reduced 2 Na:1 K stoichiometry is essential for the brine shrimp to maintain a larger Na+ gradient than other animals. NSF MCB-1515434.