“Maximal Ca2+i- regulatory site affinity of bovine heart Na+/Ca2+ exchanger requires simultaneous de-protonation and PtdIns-4,5-P2 binding”

VELIA POSADA, LUIS BEAUGÉ AND GRACIELA ELSO DE BERBERIÁN.

BIOLOGICAL CHEMISTRY

Walter de Gruyte

Lugar: Berlin; Año: 2006 vol. 388 p. 281 - 288

1431-6730

MAXIMAL Ca2+i STIMULATION OF CARDIAC Na+/Ca2+ EXCHANGE REQUIRES SIMULTANEOUS ALKALINIZATION AND BINDING OF PtdIns-4,5-P2 TO THE EXCHANGER Velia Posada ‡, Luis Beaugé and Graciela Berberián* Laboratorio de Biofísica, Instituto de Investigación Médica "Mercedes y Martín Ferreyra" (INIMEC-CONICET), Casilla de Correo 389, 5000 Córdoba, Argentina. Short title: PtdIns-4,5-P2, H+ and Ca2+i affinity of NCX1 *Correspondent author: Graciela Berberián (Email: gelso@immf.uncor.edu). Laboratorio de Biofísica, Instituto de Investigación Médica "Mercedes y Martín Ferreyra" (INIMEC-CONICET), Casilla de Correo 389, 5000 Córdoba, Argentina. Phone: (54-351) 4681465; FAX: (54-351) 4695163. Abstract By using bovine heart sarcolemmal vesicles we explored the effects of protons and PtdIns-4,5-P2 on the affinity of mammalian Na+/Ca2+ exchanger (NCX1) for intracellular Ca2+. By following the effects of extravesicular ligands in inside-out vesicles we were able to study their interactions with sites of the exchanger facing the intracellular medium of the cell. Two Na+-gradient-dependent fluxes were studied: Ca2+-uptake and Ca2+-release. In parallel, PtdIns-4,5-P2 binding to NCX1 was also investigated. Without MgATP (no ´de novo´ synthesis of PtdIns-4,5-P2), alkalinization increases the affinity for Ca2+ and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides are insensitive to alkalinization but become responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduces the affinity for Ca2+ev; this is only partially reverted by MgATP despite the increase in bound PtdIns-4,5-P2 to levels seen with alkalinization. Inhibition of Ca2+ uptake by increasing extravesicular [Na+] indicates that it is related to H+i and Na+i synergic inhibition on the Ca2+i regulatory site. Therefore, the affinity for Ca2+ of the NCX1 Ca2+i regulatory site is maximal when both intracellular alkalinization and an increase of PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occur simultaneously. In addition, protons influence the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/ or on the exchanger protein. Key words: Ca2+i regulatory site; Ca2+ transport; MgATP; Na+/Ca2+ countertransport; Na+i inhibition; pH; phosphoinositides.