The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics -: Implications for Ca2+ signaling
The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics -: Implications for Ca2+ signaling
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DOI：
10.1074/jbc.m701129200
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发表时间：
2007-08-31
影响因子：
4.8
通讯作者：
Strehler, Emanuel E.
中科院分区：
文献类型：
--
作者：
Caride, Ariel J.;Filoteo, Adelaida G.;Strehler, Emanuel E.
调节结构域的抑制作用以及与钙调素（CaM）的相互作用在质膜钙泵（PMCA）亚型之间存在差异。为了探索这些差异，研究了CaM对PMCA 4a的动力学影响，并与PMCA 4b的动力学影响进行了比较。钙调素激活PMCA4a的最大表观速率常数几乎是PMCA4b的两倍，而这两种亚型的激活率显示出类似的依赖于Ca 2+。通过CaM去除PMCA4a的失活也比PMCA4b更快，并且Ca2+显示出小得多的影响（2倍与30倍修饰）。确定总速率的各个步骤的速率常数从停流实验中获得，其中通过其荧光的变化观察TA-CaM的结合。TA-CaM与PMCA 4a的两种构象结合，即高活性的"开放"构象和低活性的"封闭"构象。与PMCA 4b（Penheiter，A. R.，Bajzer，Z.，Filoteo，A. G.，索罗盖特河，Torok，K.，和Caride，A. J.（2003）Biochemistry 41，12115 - 12124），PMCA 4a的模型预测闭合形式的抑制较少，而开放和闭合形式之间的平衡快得多。基于可用的动力学参数，我们确定了常数，以适应PMCA4b过表达中国仓鼠卵巢细胞中的Ca2+信号的形状。使用PMCA4a的常数，并允许其他系统的参数的微小变化，有助于Ca2+信号，然后，我们模拟的影响PMCA4a的形状的Ca2+信号在中国仓鼠卵巢细胞。结果再现了已发表的数据（Brini，M.，科莱托湖，Pierobon，N.，Kraev，N.，Guerini，D.，Carafoli，E.（2003）J.Biol.Chem.278，24500 - 24508），从而证明了改变的调节动力学对于PMCA同种型的不同功能性质的重要性。
The inhibition by the regulatory domain and the interaction with calmodulin ( CaM) vary among plasma membrane calcium pump ( PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect ( 2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaMbinds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b( Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., Torok, K., and Caride, A. J. ( 2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data ( Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. ( 2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.