Interaction of PIP₂ with the XIP region of the cardiac Na/Ca exchanger

The sarcolemmal Na/Ca exchanger undergoes an inactivation process in which exchange activity decays over several seconds following activation by the application of Na to the intracellular surface of the protein. Inactivation is eliminated by an increase in membrane phosphatidylinositol 4,5-bisphosphate (PIP₂). Inactivation is also strongly affected by mutations to a basic 20-amino acid segment of the exchanger known as the endogenous XIP region. The hypothesis that PIP₂ directly interacts with the XIP region of the exchanger was tested. First, we investigated the ability of a peptide with the same sequence as the XIP region to bind to immobilized phospholipid vesicles. ¹²⁵I-labeled XIP bound avidly to vesicles containing only a low concentration (<3%) of PIP₂. The binding was specific, in that binding was not displaced by other basic peptides. The effects of altering the sequence of XIP peptides also indicated binding specificity. Second, we examined the functional response to PIP₂ of exchangers with mutated XIP regions. Outward Na/Ca exchange currents were measured using the giant excised patch technique. The mutated exchangers either had no inactivation or accelerated inactivation. In both cases, the exchangers no longer responded to PIPs or to PIP₂ antibodies. Overall, the data indicate that the affinity of the endogenous XIP region for PIP₂ is an important determinant of the inactivation process.