A new cell-penetrating peptide that blocks the autoinhibitory XIP domain of NCX1 and enhances antiporter activity

The plasma membrane Na⁺/Ca²⁺ exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na⁺ ions for 1Ca²⁺ ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIP_NCX1), represent an autoinhibitory region involved in the Na⁺-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIP_NCX1 region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562-688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIP_NCX1 domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful.