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

Abstract

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...

Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes

“…As shown in this study, phosphorylation of PLM by PKA or PKC does not influence the effect of PLM on the K ϩ affinity of Na,K-ATPase ␣1/␤1 and ␣2/␤1 isozymes. The lack of an effect of PLM phosphorylation on the apparent K ϩ affinity of Na,K-ATPase is consistent with our previous observation that the effect of several FXYD proteins on the K ϩ affinity is mainly determined by interactions in the transmembrane domains of the two proteins (38,39), which may not be influenced by modifications in the cytoplasmic domain of PLM.…”

Structural and functional interactions between FXYD5 and the Na+-K+-ATPase

“…It was further demonstrated that three transmembrane residues are particularly important for the physical interaction of FXYD5 with the pump. Taken together with previous data on other FXYD proteins (23,24,26), it appears that different FXYD proteins interact similarly with the Na ϩ -K ϩ -ATPase and their different functional effects are likely to stem from a small number of key residues.…”

Interaction of Mat-8 (FXYD-3) with Na+/K+-ATPase in Colorectal Cancer Cells

“…33) Two-thirds of the face of the transmembrane helix in FXYD-7, which contains the conserved two Gly residues, was suggested to be important for both structural and functional interaction with the Na ϩ /K ϩ -ATPase a subunit. 34) Since a buried Gly residue has the smallest average volume, 35) these conserved Gly residues would form a hollow for suitable interaction between transmembrane helices of integral membrane proteins.…”