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

Phosphatidylinositol biphosphate (PtdIns-4,5P(2)) 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-gamma-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtcIns-4,5P(2) incubated in the absence of MgATP and at normal [Na+](i), alkalinization increases the affinity for Ca-i(2+), 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 Ca-i(2+) 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-4,5P(2). Experiments on NCX1-PtdIns-4,5P(2) cross-coimmunoprecipitation show that the relevant PtdIns-4,5P(2) is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca-i(2+) regulatory site is seen in the deprotonated and PtdIns-4,5P(2)-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. (C) 2009 Elsevier Ltd. All rights reserved.