In most animal cells, the Na/K pump maintains the electrochemical gradient for Na+ across the plasmalemma by extruding 3 Na+ and importing 2 K+ per ATP molecule hydrolyzed. Brine shrimp (Artemia franciscana) adapted to high salinity express a Na/K pump α subunit that has two Asn-to-Lys substitutions within the normally conserved ion-binding region (1). To address the molecular mechanisms by which these substitutions are advantageous, we expressed Xenopus α1/β3 pumps in Xenopus oocytes and used two-electrode voltage clamp (TEVC) and giant inside-out patches to evaluate the functional effect of introducing these substitutions (N333K and N785K), individually and concurrently. TEVC was performed on Na+-loaded oocytes to study external ion binding. The K0.5 for activation of Na/K pump current by external K+ (K+o) at 0 mV, without Na+o, was (in mM): 0.21±0.03 (n=11) for wild type (WT), 0.23 ±0.05 (n=6) for N333K, 4.06 ±1.4 (n=8) for N785K and 0.32 ±0.08 (n=7) for N333K/N785K. In the presence of 125 mM Na+o, without K+o, Na/K pumps undergo voltage-dependent transitions between Na+-bound and Na+-free states that produce transient currents. Voltage dependence of the charge (Q) moved in these transitions follows a Boltzmann distribution where the center (V1/2) relates to the Na/K pump’s affinity for Na+o (~25 mV shift per 2-fold affinity change (2)). The V1/2 of these Q-V curves was (in mV): -46 ±1.3 (n=9) for WT, -123 ±9 for N333K (n=13), -120 ±2 (n=8) for N785K and -92 ±1 (n=8) for N333K/N785K, indicating a reduction in Na+o affinity; ~10-fold in single mutants and ~4-fold in N333K/N785K. Measurement of the mutants’ apparent affinity for intracellular Na+, by following the [Na+i] dependence of total Q moved in inside-out patches as previously described (3), is underway. Non-additive effects of the mutations demonstrate coupling between these residues. Molecular dynamics simulations (150 ns) of the mutations in ion-bound crystal structures explain why the two mutations occur concurrently. In particular, the reduced ion affinity induced by K785 is “rescued” by the presence of K333, as the latter repels K785, forcing it inside the ion-binding pocket where it substitutes one Na+ or K+. Indeed, simulations of N333K/N785K with one ion removed from the structures increased ion-binding site stability, consistent with a proposed altered stoichiometry. To determine the stoichiometry of N333K/N785K we measured 86Rb+ (K+ congener) uptake in oocytes under TEVC and found that , as expected, wild-type pumps imported 2.11 ± 0.07 (n=40) Rb+ per charge extruded, while N333K/N785K imported 1.01 ± 0.05 (n=13) Rb+ per charge. Thus, this reduced 2 Na+:1 K+:1ATP stoichiometry enables the shrimp to maintain a much larger Na+ gradient than other animals.