Phosphatidyl inositol-4,5-bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes

Abstract

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100-130% when membrane vesicles are incubated (15-20 s at 37 degrees C) with 1 mM MgATP and 1 microM Ca2+ (b) in the presence of 100 microM Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 microM Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.