Regulation of phosphatidylinositol-4,5-biphosphate bound to the bovine cardiac Na+/Ca2+ exchanger.
Western blot and cross immunoprecipitation analysis with specific antibodies demonstrate that in bovine heart sarcolemmal vesicles phosphatidylinositol-4,5-biphosphate (PtdIns-4,5-P(2)) binds strongly to the Na(+)/Ca(2+) exchanger (NCX1). This binding is modulated by ATP, Ca(2+), vanadate, exchanger inhibitory peptide (XIP), and PLC-PtdIns specific in a way resembling the ATP regulation of the exchange fluxes. With 1 microM Ca(2+), 3 mM Mg(2+), and 0.4 mM vanadate, 1 mM ATP increased about twofold the bound PtdIns-4,5-P(2), reaching a steady state in 3-5 s at 37 degrees C. With 100 microM Ca(2+), ATP had no effect on the PtdIns-4,5-P(2) bound to NCX1 or on the exchange fluxes. Without vanadate the bound PtdIns-4,5-P(2) was largely reduced; under this condition ATP failed to increase it and did not stimulate the exchanger. XIP inhibits the exchanger, more noticeable in the absence of ATP. With XIP, ATP does not modify the levels of bound PtdIns-4,5-P(2); however there is a small but distinct ATP stimulation of the exchanger. Vesicles pretreated with PtdIns-PLC, showed no de novo, [(32)P]ATP-induced, production of PtdIns-4,5-P(2), but some ATP-stimulated increase in the bound PtdIns-4,5-P(2) was detected; however, that increase did not exceed the levels found with vanadate and no ATP. These results indicate that in bovine heart sarcolemmal vesicles, ATP upregulation of NCX1 is related to PtdIns-4,5-P(2) bound to the exchanger, perhaps over a "threshold" or "unspecific" amount. In addition, vanadate could influence the amount of detected PtdIns-4,5-P(2) either by inhibiting phosphoinositide-specific phosphatases and/or by inducing a redistribution of PtdIns-4,5-P(2) molecules associated with the Na(+)/Ca(2+) exchanger.
Beaugé
Luis
L
Laboratorio de Biofísica, Instituto de Investigación Médica Mercedes y Martín Ferreyra, 5000 Córdoba, Argentina. lbeauge@immf.uncor.edu
Asteggiano
Carla
C
Berberián
Graciela
G
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding; D019831:Sodium-Calcium Exchanger / Q000187:drug effects / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C066745:exchanger inhibitory peptide; D014638:Vanadates; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases