The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the gamma subunit, has been shown to decrease the apparent Na+ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of gamma mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of gamma effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing alphabeta pumps modulates Na+ affinity in a manner similar to transfected full-length gamma subunit. Using mutated gamma peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system.