Urbanczyk Jason, Chernysh Olga, Condrescu Madalina, Reeves John P
Department of Pharmacology & Physiology, UMDNJ - NJ Medical School, 185 South Orange Avenue, PO Box 1709, Newark, NJ 07101-1709, USA.
J Physiol. 2006 Sep 15;575(Pt 3):693-705. doi: 10.1113/jphysiol.2006.113910. Epub 2006 Jun 29.
The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.3 to > 1.8 microm. Chinese hamster ovary cells expressing the D447V exchanger showed little or no activity under physiological ionic conditions unless cytosolic [Ca(2+)] was elevated to > 1 microm. However, when cytosolic [Na(+)] was increased to 20 mm or more (using ouabain-induced inhibition of the Na(+),K(+)-ATPase or the ionophore gramicidin), cells expressing the D447V mutant rapidly accumulated Ca(2+) or Ba(2+) when the reverse (Ca(2+) influx) mode of NCX activity was initiated, although initial cytosolic [Ca(2+)] was < 100 nm. Importantly, the time course of Ca(2+) uptake did not display the lag phase that reflects allosteric Ca(2+) activation of NCX activity in the wild-type NCX1.1; indeed, at elevated [Na(+)], the D447V mutant behaved similarly to the constitutively active deletion mutant Delta(241-680), which lacks the regulatory Ca(2+) binding sites. In cells expressing wild-type NCX1.1, increasing concentrations of cytosolic Na(+) led to a progressive shortening of the lag phase for Ca(2+) uptake. The effects of elevated [Na(+)] developed rapidly and were fully reversible. The activity of the D447V mutant was markedly inhibited when phosphatidylinositol 4,5-bisphosphate (PIP2) levels were reduced. We conclude that when PIP2 levels are high, elevated cytosolic [Na(+)] induces a mode of exchange activity that does not require allosteric Ca(2+) activation.
心脏钠钙交换体（NCX1.1）的活性受Ca²⁺变构调节，Ca²⁺与NCX蛋白胞质侧中央亲水区的两个酸性区域结合。调节性Ca²⁺结合区域之一的突变（D447V）使变构Ca²⁺激活的半激活常数（K（h））从约0.3增加到>1.8 μmol/L。表达D447V交换体的中国仓鼠卵巢细胞在生理离子条件下几乎没有活性，除非胞质[Ca²⁺]升高到>1 μmol/L。然而，当胞质[Na⁺]增加到20 mmol/L或更高时（使用哇巴因诱导抑制钠钾ATP酶或离子载体短杆菌肽），当启动NCX活性的反向（Ca²⁺内流）模式时，表达D447V突变体的细胞迅速积累Ca²⁺或Ba²⁺，尽管初始胞质[Ca²⁺]<100 nmol/L。重要的是，Ca²⁺摄取的时间进程没有显示出反映野生型NCX1.1中NCX活性变构Ca²⁺激活的延迟期；实际上，在[Na⁺]升高时，D447V突变体的行为类似于缺乏调节性Ca²⁺结合位点的组成型活性缺失突变体Delta(241 - 680)。在表达野生型NCX1.1的细胞中，胞质Na⁺浓度增加导致Ca²⁺摄取延迟期逐渐缩短。[Na⁺]升高的影响迅速出现且完全可逆。当磷脂酰肌醇4,5 - 二磷酸（PIP2）水平降低时，D447V突变体的活性受到明显抑制。我们得出结论，当PIP2水平较高时，胞质[Na⁺]升高会诱导一种不需要变构Ca²⁺激活的交换活性模式。
