Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

摘要

NCX1 (Na+/Ca2+ exchanger 1) is an important regulator of intracellular Ca2+ 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 (pSer68-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 pSer68-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 pSer68-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 Ser68.* CBD, : calcium-binding domain; CLD, : catenin-like domain; DMEM, : Dulbecco's modified Eagle's medium; GAPDH, : glyceraldehyde-3-phosphate dehydrogenase; HF, : heart failure; NCX, : Na+/Ca2+ exchanger; HRP, : horseradish peroxidase; LV, : left ventricle; NKA, : Na+/K+-ATPase; PKC, : protein kinase C; PLM, : phospholemman; SLC8, : solute carrier 8; TBS-T, : TBS with 0.1% Tween 20; TF, : trigger factor; TM, : transmembrane segment; XIP, : exchanger inhibitory peptide