title: Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1).
abstract: NCX1 (Na(+)/Ca(2+) exchanger 1) is an important regulator of intracellular Ca(2+) and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer(68)-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer(68)-PLM-NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1-PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer(68)-PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser(68).
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Pimthanya
authors.initials: P
authors.lastname: Wanichawan
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Kjetil
authors.initials: K
authors.lastname: Hodne
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Tandekile Lubelwana
authors.initials: TL
authors.lastname: Hafver
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Marianne
authors.initials: M
authors.lastname: Lunde
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Marita
authors.initials: M
authors.lastname: Martinsen
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: William Edward
authors.initials: WE
authors.lastname: Louch
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Ole Mathias
authors.initials: OM
authors.lastname: Sejersted
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway c.r.carlson@medisin.uio.no.
authors.forename: Cathrine Rein
authors.initials: CR
authors.lastname: Carlson
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000494:pharmacology*; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman
keywords: brain; heart; peptide array; phospholemman; sodium/calcium exchanger