Abstract
Introduction: The cardiac Na+/Ca2+ exchanger 1 (NCX1) modulates excitation-contraction coupling and contributes to Ca2+ removal in cardiomyocytes. Altered expression and activity of NCX1 is linked to dysfunctional Ca2+ handling in chronic heart disease. Consequently, modulation of NCX1 activity is proposed as a therapeutic target. The cytosolic loop of NCX1, ∼550 amino acids, comprises several important docking and regulation sites. In particular, phospholemman (PLM) has been shown to interact and inhibit NCX1 activity when phosphorylated at serine 68 (pSer68-PLM). Importantly, pSer68-PLM has been shown as a substrate for protein phosphatase 1 (PP1) in the context of PLM regulation of the sodium-potassium pump. PP1 regulation of NCX1 is unknown.Hypothesis: PP1 binds to NCX1 and regulates its activity by dephosphorylating pSer68-PLM.Methods and Results: Using co-immunoprecipitation in rat heart lysates we have shown that NCX1 exists in a macromolecular complex with PP1 and PLM. This facilitates specific control of NCX function. Bioinformatic analysis revealed three putative PP1 binding sites on NCX1. Co-localization studies, co-immunoprecipitations, pull down, mutation- and peptide overlay assays indicated that PP1 bound directly to the consensus sequence R/KVxF in calcium binding domain 1 (CBD1) of NCX1. The reciprocal NCX1 binding site in PP1 was identified within residues 235-260, a region which harbours important anchoring sites. A peptide docking model was generated showing how the R/KVxF peptide may bind in the hydrophobic pocket of PP1. Surface plasmon resonance analysis indicated that the NCX1-PP1 binding is strong and stable. This binding does not inhibit PP1 activity. Co-expression of NCX1 with PLM and PP1 in HEK293 down regulates pSer68-PLM, indicating that PP1-R/KVxF- binding is a prerequisite for dephosphorylation of pSer68-PLM.Conclusion: R/KVxF motif in NCX1-CBD1 anchors PP1, does not change the activity of the enzyme and facilitates dephosphorylation of pSer68-PLM.
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