Identification of Phospholemman Residues Critical to Phospholemman Oligomerization and Na Pump Association

Abstract

Phospholemman (PLM or FXYD1) associates with and modulates Na pump (NKA) function in a manner similar to phospholamban (PLB) regulation of SERCA. That is, PLM inhibits NKA by reducing its [Na]i-affinity and PLM phosphorylation relieves this inhibition. We have also shown that PLM forms homo-oligomers (as does PLB) but it is unknown how PLM-PLM interactions occur or what functional role PLM oligomers have. Here we use site-directed mutagenesis and FRET to identify which PLM residues are critical to the PLM-NKA and PLM-PLM association, respectively. We hypothesize that mutations affecting PLM-PLM affinity will enhance PLM-NKA complexes and display stronger NKA inhibition, and vice versa. Based on crystal NKA-FXYD structure, the PLM sites G20, F28 & G31 were selected as potential NKA interaction sites and I26, I29, L30, L33&L36 as putative PLM interaction sites (analogous to leucine zippers thought to mediate PLB pentamerization). Alanine substitution of F28 eliminated PLM-NKA FRET (Fdonor increase by only 0.2±4% vs 18±2% for WT). Despite only minor effects on PLM-NKA interaction for G31A (Fdonor increase by 14±1%), its PLM-PLM FRET was significantly increased (Fdonor increase by 55±3% vs 37±5% for WT). Alanine substitution of I26, L30 & L33 all significantly reduced PLM-PLM FRET (Fdonor increase by 15±2%, 13±3% and 20±3% respectively vs 37±5% for WT) and opposing effects on PLM-NKA FRET were observed (e.g. Fdonor increases by 33±6% for L33A vs 18±2% for WT). We conclude that PLM homo-oligomers have a structural basis that parallels PLB (Leucine zipper). Moreover our hypothesis that altering the PLM affinity for NKA also changes the interaction of PLM with other PLM molecules (and vice versa) seems to be correct. Our data support the existence of a dynamic equilibrium of PLM oligomers and PLM-NKA that exerts functional regulation of NKA.