The intracellular region of FXYD1 is sufficient to regulate cardiac Na/K ATPase

FXYD1 is a transmembrane protein predominantly expressed in excitable tissues that associates with and regulates Na/K ATPase. PKA phosphorylates FXYD1 at serine 68 ( S68), however, the effects of phosphorylation on Na/K ATPase activity are not fully characterized. The objectives of this study were to characterize Na/K ATPase currents in FXYD1 wild-type (WT) and knockout ( KO) adult mouse ventricular myocytes, and investigate the effects of FXYD1 on Na/ K ATPase currents using the whole-cell patch-clamp technique. A peptide representing the 19 C-terminal residues of FXYD1 ( FXYD1(54-72)) was introduced into the interior of FXYD1 KO and WT myocytes through the patch pipette. K-sensitive Na/K ATPase currents were higher in KO myocytes ( 2.9 +/- 0.1 pA/pF; n = 4) compared with WT ( 1.9 +/- 0.1 pA/pF; n = 4). Unphosphorylated FXYD1(54-72), at a concentration of 4 mu M, reduced the currents in WT ( from 2.1 +/- 0.1 to 1.3 +/- 0.1 pA/pF; P < 0.05, n = 7) and KO ( from 2.9 +/- 0.1 to 1.7 + 0.1 pA/ pF; P < 0.05, n = 5), whereas, 1 mu M of FXYD1(54-72) phosphorylated at S68 increased currents in WT ( from 1.91 +/- 0.09 to 3.1 +/- 0.5 pA/ pF; P < 0.05, n = 6) and KO ( from 2.7 +/- 0.11 to 3.8 +/- 0.2 pA/ pF; P < 0.05, n = 6) myocytes. Coimmunoprecipitation studies demonstrated that S68 phosphorylated and unphosphorylated FXYD1(54-72) associates with Na/ K ATPase alpha 1 subunit. We conclude that unphosphorylated FXYD1 inhibits Na/ K ATPase, whereas S68 phosphorylated FXYD1 stimulates Na/ K ATPase to a level above that seen in the absence of FXYD1.