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 microM, 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 microM 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 alpha1 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.
Pavlović
Davor
D
Cardiovascular Division, The Rayne Institute, King's College London, St. Thomas Hospital, London, SE1 7EH, UK.
Fuller
William
W
Shattock
Michael J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D047468:Immunoprecipitation; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D000254:Sodium-Potassium-Exchanging ATPase