UP-REGULATION OF SQUID NERVE Na+/Ca2+ EXCHANGE BY MG.ATP: RECENT DEVELOPMENTS

: GRACIELA ELSO DE BERBERIAN; LUIS BEAUGÉ; REINALDO DIPOLO; ALBERTO PODJARNY

Simposio; Reunión Anual Sociedad Argentina de Biofísica.; 2012

Sociedad Argentina de Biofisica

In squid nerves, MgATP modulation of the Na+/Ca2+ exchanger (NCXSQ1) requires the presence of a cytosolic protein of 132 amino acids that is a member of CRABP family in the FABP superfamily. We called it ReP1-NCXSQ for regulatory protein of the NCXSQ1. ReP1-NCXSQ was cloned, expressed, and purified. The X-ray structure at 1.28 Å resolution shows the FABP- barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in E. coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the NCXSQ1 and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in this mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. Functional experiments showed that, to be active, ReP1-NCXSQ must be phosphorylated by MgATP, through the action of a kinase present in the plasma membrane. Moreover, PO4-ReP1-NCXSQ can stimulate the exchanger in the absence of ATP.  In addition, high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies identified palmitic acid as the fatty acid involved in regulation of NCXSQ1.