Posada Velia, Beaugé Luis, Berberián Graciela
Laboratorio de Biofísica, Instituto de Investigación Médica Mercedes y Martín Ferreyra, Casilla de Correo 389, 5000 Córdoba, Argentina.
Biol Chem. 2007 Mar;388(3):281-8. doi: 10.1515/BC.2007.031.
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.
我们使用牛心肌质膜囊泡研究了质子和磷脂酰肌醇 - 4,5 - 二磷酸（PtdIns - 4,5 - P2）对哺乳动物钠/钙交换体（NCX1）与细胞内钙亲和力的影响。通过追踪外翻囊泡中外囊泡配体的作用，研究了它们与面向细胞内介质的NCX1位点的相互作用。研究了两种依赖钠梯度的通量：钙摄取和钙释放。同时研究了PtdIns - 4,5 - P2与NCX1的结合情况。在没有MgATP（无法“从头”合成PtdIns - 4,5 - P2）的情况下，碱化增加了对钙的亲和力以及与NCX1结合的PtdIns - 4,5 - P2。缺乏磷酸肌醇的囊泡对碱化不敏感，但在添加外源性PtdIns - 4,5 - P2或磷脂酰肌醇加MgATP后变得有反应。酸化降低了对钙（ev）的亲和力；尽管结合的PtdIns - 4,5 - P2增加到碱化时观察到的水平，但MgATP只能部分逆转这种情况。通过增加外囊泡[Na⁺]来抑制钙摄取表明，这与H⁺（i）和Na⁺（i）对Ca²⁺（i）调节位点的协同抑制有关。因此，当细胞内碱化和与NCX1结合的PtdIns - 4,5 - P2（不仅仅是总膜PtdIns - 4,5 - P2）同时增加时，NCX1的Ca²⁺（i）调节位点对钙的亲和力最大。此外，质子影响了PtdIns - 4,5 - P2分子在周围环境和/或交换蛋白上的分布或暴露情况。
