Liberation of Pser68-Plm Inhibition of NCX1 by an Optimized Anchoring Disruptor Peptide

Abstract

The cardiac sodium-calcium exchanger (NCX1) is an important regulator of intracellular Ca2+ ([Ca2+]i) and a potential therapeutic target in heart failure. Among potential interacting partners regulating NCX1 activity is the transmembrane protein phospholemman (PLM); a substrate for protein kinase A and protein kinase C. Several reports have demonstrated that binding of phosphorylated PLM (pSer68-PLM) to NCX1 leads to NCX1 inhibition, while other studies have failed to demonstrate a functional interaction of these proteins. In present study, we aimed to analyze the biological function of the pSer68-PLM-NCX1 interaction by developing anchoring disruptor peptides. We observed that PLM co-fractionated and co-immunoprecipitated with NCX1 in rat left ventricle (LV) and in co-transfected HEK293 cells. Strong binding of pSer68-PLM to one of the previously reported NCX1-QKHPD regions was demonstrated in pull-down and overlay assays. Single amino acid substitutions of native NCX1 peptide sequence 1-KHPDKEIEQLIELANYQVLS-20 revealed that single substitutions at position 1-11 (particularly with tryptophan or tyrosine) increased binding affinity of the peptide to pSer68-PLM. Interestingly, we found that double substitution at position 4 and 6 with tyrosine (D4Y and E6Y) in the native peptide sequence enhanced the binding affinity to pSer68-PLM 8-fold, compared to native sequence. The optimized peptide 1-KHPYKYIEQLIELANYQVLS-20 was further tested in a series of electrophysiological experiments using whole-cell voltage clamp technique, with voltage-ramp protocol employed to elicit NCX current. Constitutively phosphorylated (S68D) PLM was observed to inhibit NCX1 current in both forward and reverse mode. Inclusion of the disruptor peptide in the patch pipette reversed S68D-PLM inhibition of NCX1, while NCX current was not altered by a scrambled peptide control. Taken together these data strongly suggest that PLM interacts directly with NCX1, and that when PLM is phosphorylated at serine 68, the interaction inhibits NCX1 activity.