Oligomeric interactions between phospholamban molecules regulate Ca-ATPase activity in functionally reconstituted membranes.
Oligomeric interactions between phospholamban molecules regulate Ca-ATPase activity in functionally reconstituted membranes.
复制标题
受磷蛋白分子之间的寡聚相互作用调节功能重构膜中的 Ca-ATP 酶活性。
DOI：
10.1021/bi002891t
复制
发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Bigelow,DJ
中科院分区：
文献类型：
--
作者：
Yao,Q;Chen,LT;Li,J;Brungardt,K;Squier,TC;Bigelow,DJ
受磷蛋白（Phospholamban，PLB）是心脏β-肾上腺素能级联反应的主要靶点，是Ca-ATP酶转运活性的内源性抑制剂。为了确定PLB分子之间的寡聚相互作用是否参与调节Ca-ATP酶转运活性，我们研究了PLB和Ca-ATP酶在纯化PLB的蛋白脂质体中的功能相互作用，所述蛋白脂质体与分离自心脏肌浆网（SR）的Ca-ATP酶的SERCA 2a同种型功能性共重构。该重构制剂的钙敏感性和cAMP依赖性蛋白激酶（PKA）的功能刺激与心脏SR微粒体中Ca-ATP酶的钙敏感性几乎相同，确保了该重构制剂的功能相关性。在共价修饰单个赖氨酸（即，在与Ca-ATP酶共重建之前，用异硫氰酸荧光素（FITC）测定从心脏SR膜分离的PLB中的Lys 3）。PLB的FITC修饰不干扰PLB抑制Ca-ATP酶的能力，因为与Ca-ATP酶共重构的FITC-PLB表现出与在天然SR膜中观察到的Ca-ATP酶活化类似的钙依赖性。因此，PLB与Ca-ATP酶的功能排列不被FITC修饰所修饰。使用荧光共振能量转移（FRET）近端PLB分子之间的FITC-PLB的各向异性的变化，以测量FITC发色团的平均大小和空间排列，我们发现，PLB自我关联，形成低聚物的空间排列相对于彼此是在协议与早期的建议，PLB主要存在作为homopentamer。在用FITC共价修饰后PKA不能活化PLB，这允许鉴定与Ca-ATP酶活化相关的PLB分子之间的功能性相互作用。通过PKA的Ca-ATP酶激活的二阶损失被观察到作为FITC-PLB的分数贡献的函数，表明在含有Ca-ATP酶的四元复合物内的两个PLB分子的PKA依赖性激活对于Ca-ATP酶的激活是必要的。我们认为PKA激活两个PLB分子的需要代表了一种生理机制，以确保心脏中β-肾上腺素能刺激后Ca-ATP酶的激活仅发生在PKA激活的阈值水平以上。
Phospholamban (PLB) is a major target of the β-adrenergic cascade in the heart, and functions as an endogenous inhibitor of Ca-ATPase transport activity. To identify whether oligomeric interactions between PLB molecules are involved in regulating Ca-ATPase transport activity, we have investigated functional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionally co-reconstituted with the SERCA2a isoform of the Ca-ATPase isolated from cardiac sarcoplasmic reticulum (SR). The calcium sensitivity of this reconstituted preparation and functional stimulation by cAMP-dependent protein kinase (PKA) are virtually identical to those of the Ca-ATPase in cardiac SR microsomes, ensuring the functional relevance of this reconstituted preparation. Interactions between PLB molecules were measured following covalent modification of the single lysine (i.e., Lys3) in PLB isolated from cardiac SR membranes with fluorescein isothiocyanate (FITC) prior to co-reconstitution with the Ca-ATPase. FITC modification of PLB does not interfere with the ability of PLB to inhibit the Ca-ATPase, since FITC-PLB co-reconstituted with the Ca-ATPase exhibits a similar calcium dependence of Ca-ATPase activation to that observed in native SR membranes. Thus, the functional arrangement of PLB with the Ca-ATPase is not modified by FITC modification. Using changes in the anisotropy of FITC-PLB resulting from fluorescence resonance energy transfer (FRET) between proximal PLB molecules to measure the average size and spatial arrangement of FITC chromophores, we find that PLB self-associates to form oligomers whose spatial arrangement with respect to one another is in agreement with earlier suggestions that PLB exists predominantly as a homopentamer. The inability of PKA to activate PLB following covalent modification with FITC permits functional interactions between PLB molecules associated with the Ca-ATPase activation to be identified. A second-order loss of Ca-ATPase activation by PKA is observed as a function of the fractional contribution of FITC-PLB, indicating that PKA-dependent activation of two PLB molecules within a quaternary complex containing the Ca-ATPase is necessary for activation of the Ca-ATPase. We suggest that the requirement for activation of two PLB molecules by PKA represents a physiological mechanism to ensure that activation of the Ca-ATPase following β-adrenergic stimulation in the heart only occurs above a threshold level of PKA activation.