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

Abstract

Phosphatidylinositol biphosphate (PtdIns-4,5P(2)) plays a key role in the regulation of the mammalian heart Na(+)/Ca(2+) exchanger (NCX1) by protecting the intracellular Ca(2+) 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 PtdIns-4,5P(2) incubated in the absence of MgATP and at normal [Na(+)](i), alkalinization increases the affinity for Ca(2+)(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 Ca(2+)(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-4,5P(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(2+)(i) 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.