Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na+/Ca2+ exchanger

Phosphatidylinositol biphosphate (PtdIns-4,5P2) plays a key role in the regulation of the mammalian heart Na+/Ca2+ exchanger (NCX1) by protecting the intracellular Ca2+ regulatory site against H+i and (H+i + Na+i) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P2 incubated in the absence of MgATP and at normal [Na+]i, alkalinization increases the affinity for Ca2+i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca2+i any further. On the other hand, prevention of Na+i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P2. Experiments on NCX1-PtdIns-4,5P2 cross-coimmunoprecipitation show that the relevant PtdIns-4,5P2 is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca2+i regulatory site is seen in the deprotonated and PtdIns-4,5P2-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.