Modulation of Na,K-ATPase by the γ Subunit

Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,KATPase. Mutational effects on the stable association with Na,KATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. By using the free energy from the hydrolysis of one ATP molecule, Na,K-ATPase, a ubiquitous P-type ATPase, transports three Na ϩ out of the cell in exchange for two K ϩ into the cell. The minimal functional unit of Na,K-ATPase consists of a catalytic ␣ subunit and a regulatory ␤ subunit. Four ␣ and 3 ␤ isoforms exist and are expressed in a tissuespecific manner.The major physiological role of Na,K-ATPase is to create and maintain the Na ϩ /K ϩ gradient across the cell membrane, which is important for the maintenance of cell volume and membrane potential. Moreover, the Na ϩ gradient serves as an energy source for numerous secondary transport systems. Finally, Na,K-ATPase is essential for specialized functions of various tissues. In renal epithelial cells, Na,K-ATPase is exclusively expressed at the basolateral membrane and becomes the driving force for transepithelial Na ϩ reabsorption. In nervous tissue, Na,K-ATPase restores the Na ϩ and K ϩ gradients during action potentials and thus ensures neuronal excitability. In skeletal and heart muscle, the Na ϩ gradient coupled to Na ϩ /Ca 2ϩ exchanger activity controls the intracellular Ca 2ϩ concentration, which influences muscle contractility. The activity of Na,K-ATPase is subjected to short-and long-term regulation (1) mediated by the intracellular Na ϩ concentration, neurotransmitters, and hormones. Recently, members of the FXYD family (2) have been shown to be tissue-specific regulators of the Na,K-ATPase (3). FXYD proteins are small membrane proteins that are characterized by an N-terminal FXYD motif, two highly conserved glycine residues in the TM domain, and a serine residue at the cytoplasmic end of the TM domain. FXYD7, an O-glycosylated protein, is only expressed in brain, in both neurons and glial cells (4). FXYD7 associates with ␣␤1 isozymes, but not with ␣␤2 isozymes, when expressed in Xenopus oocytes. Stable association of FXYD7 with Na,K-ATPase modifies its apparent affinity for external K ϩ by increasing by ϳ2-fold the K1 ⁄ 2 value for K ϩ over a large range of membrane potentials in the presence and absence of external Na ϩ . In addition, FXYD7 modifies the apparent affinity for extrace...

supporting

confidence: 64%

Role of the Transmembrane Domain of FXYD7 in Structural and Functional Interactions with Na,K-ATPase

Crambert

Thuillard

et al. 2005

“…Nevertheless, a clear distinction between interaction sites involved in the stable association or in a specific functional effect is not obvious in all cases. Based on experiments with mimetic peptides, it has indeed been proposed that Gly 41 (corresponding to Gly 40 in FXYD7) is not primarily involved in Na,K-ATPase interaction but rather mediates the effect on the Na ϩ kinetics of FXYD2 (34). Knowing that FXYD7 has no effect on the apparent Na ϩ affinity of Na,K-ATPase, this result is difficult to reconcile with our results which show that a G40A mutant of FXYD7, decreases the association efficiency and completely abolishes the K ϩ effect.…”

Section: Discussionmentioning

confidence: 99%

FXYD7, Mapping of Functional Sites Involved in Endoplasmic Reticulum Export, Association With and Regulation of Na,K-ATPase

Crambert

Swee

et al. 2004

The brain-specific FXYD7 is a member of the recently defined FXYD family that associates with the ␣1-␤1 Na,K-ATPase isozyme and induces an about 2-fold decrease in its apparent K ؉ affinity. By using the Xenopus oocyte as an expression system, we have investigated the role of conserved and FXYD7-specific amino acids in the cellular routing of FXYD7 and in its association with and regulation of Na,K-ATPase. In contrast to FXYD2 and FXYD4, the studies on FXYD7 show that the conserved FXYD motif in the extracytoplasmic domain is not involved in the efficient association of FXYD7 with Na,K-ATPase. On the other hand, the conserved Gly 40 and Gly 29 , located on the same face of the transmembrane helix, were found to be implicated both in the association with and the regulation of Na,K-ATPase. Mutational analysis of FXYD7-specific regions revealed the presence of an ER export signal at the end of the cytoplasmic tail. Deletion of a C-terminal valine residue in FXYD7 significantly delayed and decreased its O-glycosylation processing and retarded the rate of its cell surface expression. This result indicates that the C-terminal valine residue is involved in the rapid and selective ER export of FXYD7, which could explain the observed post-translational association of FXYD7 with Na,KATPase. In conclusion, our study on FXYD7 provides new information on structural determinants of general importance for FXYD protein action. Moreover, FXYD7 is identified as a new member of proteins with a regulated ER export, which suggests that, among FXYD proteins, FXYD7 has a particular regulatory function in brain.

“…Co-immunoprecipitation-The method used was a modification of Garty et al (27) as described elsewhere (21). Band densities were quantified using Molecular Dynamics ImageQuant software.…”

Section: Of the Lowry Methods (26)mentioning

confidence: 99%

Regions of the Catalytic α Subunit of Na,K-ATPase Important for Functional Interactions with FXYD 2

Zouzoulas¹,

Blostein

2006

Self Cite

The ␥ modulator (FXYD 2) is a member of the FXYD family of single transmembrane proteins that modulate the kinetic behavior of Na,K-ATPase. This study concerns the identification of regions in the ␣ subunit that are important for its functional interaction with ␥. An important effect of ␥ is to increase K ؉ antagonism of cytoplasmic Na ؉ activation apparent as an increase in K Na at high. We show that although ␥ associates with ␣1, ␣2, and ␣3 isoforms, it increases the K Na of ␣1 and ␣3 but not ␣2. Accordingly, chimeras of ␣1 and ␣2 were used to identify regions of ␣ critical for the increased K Na . As with ␣1 and ␣2, all chimeras associate with ␥. Kinetic analysis of ␣2 front /␣1 back chimeras indicate that the C-terminal (Lys 907 -Tyr 1018 ) region of ␣1, which includes transmembrane (TM)9 close to ␥, is important for the increase in K Na . However, similar experiments with ␣1 front /␣2 back chimeras indicate a modulatory role of the loop between TMs 7 and 8. Thus, as long as the ␣1 L7/8 loop is present, replacement of TM9 of ␣1 with that of ␣2 does not abrogate the ␥ effect on K Na . In contrast, as long as TM9 is that of ␣1, replacement of L7/8 of ␣1 with that of ␣2 does not abolish the effect. It is suggested that structural association of the TM regions of ␣ and FXYD 2 is not the sole determinant of this effect of FXYD on K Na but is subject to long range modulation by the extramembranous L7/8 loop of ␣.The Na,K-ATPase or sodium pump is an integral membrane protein found in the cells of virtually all higher eukaryotes. It couples the hydrolysis of one molecule of ATP to the electrogenic exchange of three intracellular Na ϩ for two extracellular K ϩ ions against their electrochemical gradients.The sodium pump is an oligomer of two subunits, a large catalytic ␣ subunit and a smaller, highly glycosylated ␤ subunit whose role is to ensure the proper folding, insertion, and maturation of ␣ in the plasma membrane. Four isoforms of ␣ and three of ␤ are expressed in a tissue-and development-specific manner. (for reviews, see Refs. 1 and 2). The sodium pump is subject to diverse modes of regulation by substrates, membrane-associated components, hormones, and neurotransmitters (3). In recent years, considerable attention has been focused on tissue-specific regulation by small transmembrane proteins referred to as FXYD proteins (for reviews, see Refs. 4 -7). These proteins belong to a gene family of small, single transmembrane proteins, with the exception of MAT-8 which has two transmembranes (TM(s)).3 There are at least seven members of which several (FXYDs 1-4 and 7) appear to regulate the kinetic behavior of the sodium pump in distinct and specific ways, in particular the apparent affinity of the enzyme for ligands (Na ϩ , K ϩ , and ATP). Members possess a 35-amino-acid signature sequence that includes 6 invariant amino acids before, in, and after the TM domain. The N-terminal FXYD (Phe-Xaa-Tyr-Asp) motif is invariant, along with two glycines in the TM domain, and a serine in the C-terminal domain. The first FXYD...