Abstract 21311: Peroxiredoxin 6 is Recruited to the Cardiac Sodium Pump Complex by Phospholemman
Phospholemman (FXYD1, PLM), the principal sarcolemmal substrate for protein kinases A and C (PKA, PKC) in the heart, is a regulator of the cardiac sodium (Na) pump. We investigated proteins that interact with PLM in adult rat ventricular myocytes (ARVM) using bifunctional crosslinking reagents and co-immunoprecipitation. Digitonin-permeabilized ARVM were treated with the heterobifunctional crosslinking reagent sulfo-lc-smpt, and lysates immunoblotted for PLM. We found sulfo-lc-smpt quantitatively crosslinked PLM to a 20–25kDa protein. Co-immunoprecipitation experiments indicated the crosslinked adduct was PLM linked to the anti-oxidant protein peroxiredoxin 6 (prdx6). PLM phosphorylation at serine 68 (PKA activation with 10µM forskolin) or at serines 63, 68 and threonine 69 (PKC activation with 300nM PMA) had no effect on the ability of sulfo-lc-smpt to crosslink PLM to prdx6. Hydrogen peroxide treatment of ARVM (1–100µM) was also without effect on the binding of prdx6 to PLM. The cardiac Na pump and PLM reside in caveolae in ARVM. Proteomic analysis and immunoblotting suggested that prdx6 is also present in caveolae prepared from ARVM. To assess the biochemical consequences for the Na pump of the interaction between PLM and prdx6, we investigated the ability of transfected PLM to recruit prdx6 and therefore protect the Na pump alpha-1 subunit from cysteine oxidation in HEK cells. Cells were treated with hydrogen peroxide (10 – 500µM), lysed, and reduced cysteines labelled with biotinylated iodoacetamide. We found no difference between cysteine oxidation status of the Na pump alpha-1 subunit between PLM-transfected HEK cells and control cells. In conclusion, our data suggest a new role for PLM in the heart. As well as being responsible for kinase-mediated regulation of the cardiac Na pump, it is responsible for recruitment of prdx6 to this ion transporter. Prdx6 catalyzes the reduction of hydrogen peroxide, fatty acid hydroperoxides and phospholipid hydroperoxides using glutathione. Oxidised lipids are established inhibitors of the Na pump and we therefore propose that recruitment of prdx6 by PLM may protect the local membrane environment in which the Na pump resides, and therefore protect the cardiac Na pump during periods of oxidative stress.
- © 2010 by American Heart Association, Inc.