The Na+,K+-ATPase builds and maintains the Na+ and K+ gradient across the plasmalemma. Functional Na+,K+-ATPase requires association of α- and β-subunits. A third single transmembrane protein of the FXYD family (FXYD1-FXYD7) modulate Na+,K+-ATPase function in a tissue specific manner. Here, we used electrophysiology on oocytes expressing human α1β1 Na+,K+-ATPase to evaluate the functional effects of association with human FXYD2, 4, 6 & 7. Under two-electrode voltage clamp, in the presence of 125 mM Na+o, application of K+o activated outward currents (due to 3Na+/2K+ exchange) with voltage-dependent apparent dissociation constant. For α1β1, K0.5,0 = 1.1 ±0.1 mM and K0.5100= 1.5 ±0.3 mM (at 0, n=6, and −100 mV, n=6, respectively). The K0.5,0 for all FXYDs was 1-1.2 mM; the K0.5100 was ∼1 mM for FXYD2 and FXYD6 and ∼2 mM for FXYD 4 and FXYD7. Thus, these FXYDs induced changes in K+o affinity could be relevant at hyperpolarized potentials. We measured the transient charge movement observed in the absence of K+o, which describes the voltage-dependent equilibrium between Na+-bound and externally open Na+-free states. The steady-state charge-voltage curves were fitted with a Boltzmann distribution. At constant [Na+]o a shift in the distribution's center (V1/2) to positive voltages signals an increase in Na+o affinity while a shift to negative voltages indicates a reduction, with a 25 mV per two fold change in Na+o affinity. The V1/2 were (in mV) −41 ±0.5 (α1β1, n=6), −16 ±2 (FXYD4, n=4), −26 ± 0.3 (FXYD7, n=2), −57 ±2 (FXYD2, n=7), and −60 ±3 (FXYD6, n=7). These results suggest similar increase in Na+o affinity by FXYD4 or FXYD7 and identical reduction in Na+o affinity by FXYD2 and FXYD6. Measurement of K0.5 for Na+K+-ATPase activation by intracellular Na+ in inside-out patches is underway. Supported by R15-NS081570-01A1.