A study of the membrane association and regulatory effect of the phospholemman cytoplasmic domain.
Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K(+) and Na(+) and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM(38-72)) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM(38-72) associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM(38-72) also lowers the maximal velocity of ATP hydrolysis (V(max)) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V(max). These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.
School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK.
Eleri
E
Hughes
Christopher A P
CA
Whittaker
Igor L
IL
Barsukov
Mikael
M
Esmann
David A
DA
Middleton
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002151:Calorimetry; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D012694:Serine / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
C091777:FXYD3 protein, human; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D010750:Phosphoproteins; C069141:phospholemman; D012694:Serine; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium