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Four of the seven members of the FXYD protein family have been identified as specific regulators of Na,K-ATPase. In this study, we show that FXYD3, also known as Mat-8, is able to associate with and to modify the transport properties of Na,K-ATPase. In addition to this shared function, FXYD3 displays some uncommon characteristics. First, in contrast to other FXYD proteins, which were shown to be type I membrane proteins, FXYD3 may have a second transmembrane-like domain because of the presence of a noncleavable signal peptide. Second, FXYD3 can associate with Na,K-as well as H,K-ATPases when expressed in Xenopus oocytes. However, in situ (stomach), FXYD3 is associated only with Na,KATPase because its expression is restricted to mucous cells in which H,K-ATPase is absent. Coexpressed in Xenopus oocytes, FXYD3 modulates the glycosylation processing of the ␤ subunit of X,K-ATPase dependent on the presence of the signal peptide. Finally, FXYD3 decreases both the apparent affinity for Na ؉ and K ؉ of Na,K-ATPase. INTRODUCTIONThe Na,K-ATPase is an ubiquitous enzyme consisting of an ␣ and a ␤ subunit, which is responsible for the creation and maintenance of the Na ϩ and K ϩ gradients across the cell membrane by transporting three Na ϩ out and two K ϩ into the cell. This function is crucial for cell survival and body homeostasis because the Na ϩ gradient is used as an energy source to transport ions or solutes and is at the origin of the vectorial Na ϩ reabsorption in the kidney and of action potentials in excitable tissues.Regulation of the activity and expression of Na,K-ATPase is tight and governed by a variety of mechanisms. Shortterm regulation involves protein kinases and results in modulation of the cell surface expression of the Na,K-ATPase, whereas long-term regulation, mediated by mineralocorticoid or thyroid hormone, leads to a change in the total number of Na,K-ATPase units (Therien and Blostein, 2000; for review, see Feraille and Doucet, 2001). Moreover, the existence of multiple ␣ and ␤ isoforms permits the production of isozymes with different transport properties (Crambert et al., 2000). Finally, recent experimental evidence shows that members of the FXYD protein family specifically associate with and modulate the transport properties of Na,KATPase (for review, see Crambert and Geering, 2003).The FXYD family contains seven members that share a common signature sequence encompassing the transmembrane and adjacent regions (Sweadner and Rael, 2000). So far, all characterized FXYD proteins exhibit a similar structure with a single transmembrane domain and a type I orientation that is achieved, in some, but not all cases, by the cleavage of an N-terminal signal peptide. Among the seven members (FXYD1-7), four FXYD proteins have so far been shown to regulate the Na,K-ATPase in a tissue-and isozyme-specific way. All affect the apparent affinity for extracellular K ϩ of the Na,K-ATPase, which in the case of FXYD7 (the brain-specific FXYD protein) is thought to be physiologically relevant in neuronal excitabilit...