INIMEC - CONICET   05467
INSTITUTO DE INVESTIGACION MEDICA MERCEDES Y MARTIN FERREYRA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
UP-REGULATION OF SQUID NERVE Na+/Ca2+ EXCHANGE BY MG.ATP: RECENT DEVELOPMENTS
Autor/es:
: GRACIELA ELSO DE BERBERIAN; LUIS BEAUGÉ; REINALDO DIPOLO; ALBERTO PODJARNY
Reunión:
Simposio; Reunión Anual Sociedad Argentina de Biofísica.; 2012
Institución organizadora:
Sociedad Argentina de Biofisica
Resumen:
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.