Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger.
Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na(+)/Ca(2+) exchanger (NCX1) for intracellular Ca(2+). By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na(+)-gradient-dependent fluxes were studied: Ca(2+) uptake and Ca(2+) release. PtdIns-4,5-P2 binding to NCX1 was investigated in parallel. Without MgATP (no 'de novo' synthesis of PtdIns-4,5-P2), alkalinization increased the affinity for Ca(2+) and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides were insensitive to alkalinization, but became responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduced the affinity for Ca(2+)(ev); this was only partially reversed by MgATP, despite the increase in bound PtdIns-4,5-P2 to levels observed with alkalinization. Inhibition of Ca(2+) uptake by increasing extravesicular [Na(+)] indicates that it is related to H(+)(i) and Na(+)(i) synergistic inhibition of the Ca(2+)(i) regulatory site. Therefore, the affinity of the NCX1 Ca(2+)(i) regulatory site for Ca(2+) was maximal when both intracellular alkalinization and an increase in PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occurred simultaneously. In addition, protons influenced the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/or on the exchanger protein.
Posada
Velia
V
Laboratorio de Biofísica, Instituto de Investigación Médica Mercedes y Martín Ferreyra, Casilla de Correo 389, 5000 Córdoba, Argentina.
Beaugé
Luis
L
Berberián
Graciela
G
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002126:Calcium Hydroxide / Q000737:chemistry; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*
C098975:Alkaliner; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D019831:Sodium-Calcium Exchanger; C037178:phosphatidylinositol 4-phosphate; C110374:sodium-calcium exchanger 1; D000255:Adenosine Triphosphate; D012964:Sodium; D002126:Calcium Hydroxide; D002118:Calcium