A mutation in the human FXYD2 polypeptide (Na,K-ATPase subunit ) that changes a conserved transmembrane glycine to arginine is linked to dominant renal hypomagnesemia. Xenopus oocytes injected with wild-type FXYD2 or the mutant G41R cRNAs expressed large non-selective ion currents. However, in contrast to the wild-type FXYD2 currents, inward rectifying cation currents were induced by hyperpolarization pulses in oocytes expressing the G41R mutant. Injection of EDTA into the oocyte removed inward rectification in the oocytes expressing the mutant, but did not alter the non-linear I-V relationship of the wild-type FXYD2 pseudo-steady-state currents. Extracellular divalent ions, Ca²⁺ and Ba²⁺, and trivalent cations, La³⁺ blocked both the wild-type and mutant FXYD2 currents. Site-directed mutagenesis of G41 demonstrated that a positive charge at this site is required for the inward rectification. When the wild-type FXYD2 was expressed in Madin-Darby canine kidney (MDCK) cells, the cells in the presence of a large apical to basolateral Mg²⁺ gradient and at negative potentials, had an increase in transepithelial current when compared to cells expressing the G41R mutant or control transfected cells. Moreover, this current was inhibited by extracellular Ba²⁺ at the basolateral surface. These results suggest that FXYD2 can mediate basolateral extrusion of magnesium from cultured renal epithelial cells and provides new insights into the understanding of the possible physiological roles of FXYD2 wild-type and mutant proteins.