Na+/ Ca2+ and Na+/Ca2+-K+ exchangers: biochemical and functional characterization in human platelets

BISTUÉ MILLÓN, MARÍA BEATRIZ(1); ELSO ? BERBERIAN, GRACIELA(2,4); ASTEGGIANO, CARLA GABRIELA(1,3,4)

Mendoza

Congreso; SAIB 48th Annual Meeting Argentine Society for Biochemistry and Molecular Biology XLVII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

Argentine Society for Biochemistry and Molecular Biology

Congenital Disorders of Glycosylation (CDG) are human genetic diseases due to defects in the synthesis of N-, O-glycoproteins, and even in the synthesis of glycolipids. The clinical features range from a severe multisystem to mild phenotype. Thrombus-hemorrhagic events are frequently observed in these patients. In platelets tight regulation of Ca2+ signaling is necessary to prevent inappropriate thrombus formation. The Na+/ Ca2+ (NCX) and Na+/Ca2+-K+ (NCKX) exchangers play a crucial role in controlling cytosolic [Ca2+]. The aim of this work is to contribute to the characterization of these exchangers in a human megakaryocytic cell line (DAMI cells) and human platelets. Our results demonstrate (i) the presence of NCX and the NCKX proteins in DAMI cells and human platelets by western blot, (ii) that both types of exchangers are functional by measurements of the Ca2+ (45Ca) uptake, (iii) 2 likely N-glycosylation sites in NCX protein and different N- and O-glycosylation sites for NCKX1 in the hydrophilic N-terminal segment by bio-informatics analysis, and (iv) that exchanger proteins are heavily glycosylated by lectin affinity chromatography and immune-precipitation analysis. Future studies will elucidate the functional role of glycosylation in the targeting and activity of NCX and NCKX exchangers in human platelets and CDG patients. CONICET (GI11220090100063) / FONCyT PICT2824.