INIMEC - CONICET   05467
INSTITUTO DE INVESTIGACION MEDICA MERCEDES Y MARTIN FERREYRA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
A PtdIns4P,5-kinase-NCX1 complex is essential for proper metabolic regulation of NCX1
Autor/es:
D. FORCATO,L. POSADA, L. BEAUGÉ AND G. ELSO DE BERBERIÁN.
Lugar:
Lacco Ameno, Ischia Islnd, Naples
Reunión:
Conferencia; 6ª International Conference on Na+/Ca2+ Exchange; 2011
Institución organizadora:
Naples University
Resumen:
The mammalian heart Na+/Ca2+ exchanger (NCX1) is inhibited by intracellular H+ and Na+ in a way that impairs the binding of cytosolic Ca2+ to its regulatory site and that inhibition is antagonized by MgATP through the production of PtdIns-4,5-P2. Experiments on NCX1-PtdIns-4,5-P2 co-immunoprecipitation show that the relevant PtdIns-4,5-P2 is not the overall membrane component but specifically that tightly attached to NCX1. In consonance with these results a functionally competent PtdIns-5-Kinase co-immunoprecipitates with NCX1 (Berberián et. al, 2009, Cell Calcium, 45:546-553). In this study, by using inside-out bovine heart sarcolemmal vesicles (IOV) we measured Na+i-dependent Ca2+, the  uptake, PtdIns4P,5-kinase activity and the co-immmunoprecipitation of NCX1 with PtdIns4P,5-kinase. The conditions were: native IOV and IOV pretreated with p-chloromercuribenzenesulfonate (PCMBS) or n-ethylmaleimide (NEM), alone or in combination with dithiothreitol (DTT). The experiments showed: (i) PCMBS and low [NEM] inhibit both the total and the NCX1-associated PtdIns4P,5-kinase; (ii) DTT reverses PCMBS and NEM inhibition of PtdIns4P,5-kinase; (iii)  PtdIns4P,5-kinase and NCX1 form a supramolecular complex; (iv) the formation of that complex is prevented by pretreatment with DTT; (v) DTT prevents stimulation of NCX1 at limiting Ca2+i and MgATP concentrations. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be functionally competent, and must be located in the NCX1 microenvironment closely associated to the exchanger. Suported by  Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2010-2012: GI11220090100063), Argentina.